Lung growth factor variant

ABSTRACT

The present invention provides a human lung growth factor variant (LGFV) and polynucleotides which identify and encode LGFV. The invention also provides genetically engineered expression vectors and host cells comprising the nucleic acid sequences encoding LGFV and a method for producing LGFV. The invention also provides for agonists, antibodies, or antagonists specifically binding LGFV, and their use, in the prevention and treatment of diseases associated with expression of LGFV. Additionally, the invention provides for the use of antisense molecules to polynucleotides encoding LGFV for the treatment of diseases associated with the expression of LGFV. The invention also provides diagnostic assays which utilize the polynucleotide, or fragments or the complement thereof, and antibodies specifically binding LGFV.

[0001] This application is a continuation application of U.S.application Ser. No. 09/207,980, filed Dec. 7, 1998, which is adivisional application of U.S. application Ser. No. 08,760,745, filedDec. 5, 1996, now U.S. Pat. No. 5,972,658, issued Oct. 26, 1999, bothentitled LUNG GROWTH FACTOR VARIANT, all of which applications andpatents are hereby incorporated herein by reference.

FIELD OF THE INVENTION

[0002] This invention relates to nucleic acid and amino acid sequencesof a lung growth factor variant and to the use of these sequences in thediagnosis, prevention, and treatment of various infections,spermatogenesis, autoimmune disorders, vascular diseases, and cancers.

BACKGROUND OF THE INVENTION

[0003] Hepatoma derived growth factors (HDGF) have been cloned from manand mouse. When they bind to specific membrane receptors, they triggerintracellular signaling cascades. Some of these signaling events controlthe activities of transcription factors. The unregulated activity oftranscription factors which govern cell proliferation may result in thegrowth and development of cancerous cells or in the release anddifferentiation of excess leukocytes.

[0004] Nakamura et al. (1994; J. Biol. Chem. 269:25143-49) described thehuman HDGF molecule as a monomeric, cytoplasmic peptide of 240 aminoacids and approximately 25 kD which lacks a signal sequence and bindsheparin. Northern analysis revealed that HDGF is expressed in severaltumor derived cell lines and in heart, brain, placenta, lung, liver,skeletal muscle, kidney and pancreas. Based on its homology to highmobility group proteins (HMGs), location in the cytoplasm, andassociation with cellular proliferation in both normal and tumor cells,Nakamura et al (supra) predict that HDGF is a transcription factor whichshows growth stimulating activity.

[0005] Baxevanis and Landsman (1995; Nucleic Acids Res 23:1604-13)aligned 121 HMG basic domains and reported that they fall into twogroups, the HMG½ proteins with DNA binding ability and non-canonicalHMGs, some of which are known to function as transcription factors. HMG½proteins bind and alter the structure of DNA, making the molecule moreamenable to the enzymes which carry out transcription. Support for thisrole comes from the fact that the half life of HMG½ proteins directlyparallels the S phase of the cell cycle (Morton, R. L. et al. (1996)Biochem. Biophys Res Commun. 222:268-373). Other studies have shown thatsome HMGs possess a calmodulin binding domain (Harley, V. R. et al.(1996) FEBS Lett 391:24-28) and others associate with either hormonereceptors as they bind DNA (Onate, S. A. et al. (1994) Mol Cell Biol14:3376-91) or T-cell receptors as they regulate lymphocyte geneexpression. These proteins enhance gene expression in the cells andtissues in which they are present.

[0006] HMGs have also been studied to determine their role intransforming fibroblasts and hematopoietic cells, in Burkitt's lymphoma(Morton, et al. supra), and in gastrointestinal carcinomas.

[0007] The discovery of polynucleotides encoding a lung growth factorvariant, and the variant itself, provides a means to investigate cellproliferation under normal and disease conditions. Such cytokines orgrowth factor-like molecules related to hepatoma growth factor satisfy aneed in the art by providing new diagnostic or therapeutic compositionsuseful in diagnosing and treating infections; autoimmune disorders,vascular diseases and cancers.

SUMMARY OF THE INVENTION

[0008] The present invention features a novel lung growth factor varianthereinafter designated LGFV and characterized as having 75% identityover the first 196 nucleotides and 90% identity overall to hepatomaderived growth factor.

[0009] Accordingly, the invention features a substantially purified LGFVhaving the amino acid sequence shown in SEQ ID NO: 1.

[0010] One aspect of the invention features isolated and substantiallypurified polynucleotides that encode LGFV. In a particular aspect, thepolynucleotide is the nucleotide sequence of SEQ ID NO:2.

[0011] The invention also relates to a polynucleotide sequencecomprising the complement of SEQ ID NO:2 or variants thereof. Inaddition, the invention features polynucleotide sequences whichhybridize under stringent conditions to SEQ ID NO:2.

[0012] The invention additionally features nucleic acid sequencesencoding polypeptides, oligonucleotides, peptide nucleic acids (PNA),fragments, portions or antisense molecules thereof, and expressionvectors and host cells comprising polynucleotides that encode LGFV. Thepresent invention also features antibodies which bind specifically toLGFV, and pharmaceutical compositions comprising substantially purifiedLGFV. The invention also features the use of agonists and antagonists ofLGFV.

BRIEF DESCRIPTION OF THE FIGURES

[0013]FIGS. 1A, 1B, and 1C show the amino acid sequence (SEQ ID NO:1)and nucleic acid sequence (SEQ ID NO:2) of LGFV. The alignment wasproduced using MACDNASIS PRO™ software (Hitachi Software EngineeringCo., Ltd., San Bruno, Calif.).

[0014]FIG. 2 shows the amino acid sequence alignments among LGFV (SEQ IDNO:1), and hepatoma growth factors from man and mouse (GI 598956; SEQ IDNO:3 and GI 945419; SEQ ID NO:5, respectively). The alignment wasproduced using the multisequence alignment program of DNASTAR™ software(DNASTAR Inc, Madison Wis.).

[0015]FIGS. 3A, 3B, and 3C show the nucleotide sequence alignments amongLGFV (SEQ ID NO:2), and human hepatoma growth factor (GI 598956; SEQ IDNO:4).

[0016]FIGS. 4A and 4B show the hydrophobicity plots (MACDNASIS PROsoftware) for LGFV (SEQ ID NO:1) and hepatoma growth factor (GI 598956;SEQ ID NO:3); the positive X axis reflects amino acid position, and thenegative Y axis, hydrophobicity.

[0017]FIGS. 5A, 5B, 5C, 5D, and 5E show the northern analysis of LGFVproduced using the LIFESEQ database (Incyte Pharmaceuticals Inc., PaloAlto, Calif.)

DESCRIPTION OF THE INVENTION

[0018] Before the present proteins, nucleotide sequences, and methodsare described, it is understood that this invention is not limited tothe particular methodology, protocols, cell lines, vectors, and reagentsdescribed as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

[0019] It must be noted that as used herein and in the appended claims,the singular forms “a”, “an”, and “the” include plural reference unlessthe context clearly dictates otherwise. Thus, for example, reference to“a host cell” includes a plurality of such host cells, reference to the“antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

[0020] Unless defined otherwise, all technical and scientific terms usedherein have the same meanings as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods, devices, and materials are now described. All publicationsmentioned herein are incorporated herein by reference for the purpose ofdescribing and disclosing the cell lines, vectors, and methodologieswhich are reported in the publications which might be used in connectionwith the invention. Nothing herein is to be construed as an admissionthat the invention is not entitled to antedate such disclosure by virtueof prior invention.

[0021] Definitions

[0022] “Nucleic acid sequence” as used herein refers to anoligonucleotide, nucleotide, or polynucleotide, and fragments orportions thereof, and to DNA or RNA of genomic or synthetic origin whichmay be single- or double-stranded, and represent the sense or antisensestrand. Similarly, “amino acid sequence” as used herein refers to anoligopeptide, peptide, polypeptide, or protein sequence, and fragmentsor portions thereof, and to naturally occurring or synthetic molecules.

[0023] Where “amino acid sequence” is recited herein to refer to anamino acid sequence of a naturally occurring protein molecule, “aminoacid sequence” and like terms, such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

[0024] “Peptide nucleic acid”, as used herein, refers to a moleculewhich comprises an oligomer to which an amino acid residue, such aslysine, and an amino group have been added. These small molecules, alsodesignated anti-gene agents, stop transcript elongation by binding totheir complementary strand of nucleic acid (Nielsen, P. E. et al. (1993)Anticancer Drug Des. 8:53-63)

[0025] LGFV, as used herein, refers to the amino acid sequences ofsubstantially purified LGFV obtained from any species, particularlymammalian, including bovine, ovine, porcine, murine, equine, andpreferably human, from any source whether natural, synthetic,semi-synthetic, or recombinant.

[0026] “Consensus”, as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, or which has beenextended using XL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte clone using the GELVIEW™Fragment Assembly system (GCG, Madison, Wis.), or which has been bothextended and assembled.

[0027] A “variant” of LGFV, as used herein, refers to an amino acidsequence that is altered by one or more amino acids. The variant mayhave “conservative” changes, wherein a substituted amino acid hassimilar structural or chemical properties, e.g., replacement of leucinewith isoleucine. More rarely, a variant may have “nonconservative”changes, e.g., replacement of a glycine with a tryptophan. Similar minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

[0028] A “deletion”, as used herein, refers to a change in either aminoacid or nucleotide sequence in which one or more amino acid ornucleotide residues, respectively, are absent.

[0029] An “insertion” or “addition”, as used herein, refers to a changein an amino acid or nucleotide sequence resulting in the addition of oneor more amino acid or nucleotide residues, respectively, as compared tothe naturally occurring molecule.

[0030] A “substitution”, as used herein, refers to the replacement ofone or more amino acids or nucleotides by different amino acids ornucleotides, respectively.

[0031] The term “biologically active”, as used herein, refers to aprotein having structural, regulatory, or biochemical functions of anaturally occurring molecule. Likewise, “immunologically active” refersto the capability of the natural, recombinant, or synthetic LGFV, or anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

[0032] The term “agonist”, as used herein, refers to a molecule which,when bound to LGFV, causes a change in LGFV which modulates the activityof LGFV. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to LGFV.

[0033] The terms “antagonist” or “inhibitor”, as used herein, refer to amolecule which, when bound to LGFV, blocks or modulates the biologicalor immunological activity of LGFV. Antagonists and inhibitors mayinclude proteins, nucleic acids, carbohydrates, or any other moleculeswhich bind to LGFV.

[0034] The term “modulate”, as used herein, refers to a change or analteration in the biological activity of LGFV. Modulation may be anincrease or a decrease in protein activity, a change in bindingcharacteristics, or any other change in the biological, functional orimmunological properties of LGFV.

[0035] The term “mimetic”, as used herein, refers to a molecule, thestructure of which is developed from knowledge of the structure of LGFVor portions thereof and, as such, is able to effect some or all of theactions of cytokine-like molecules.

[0036] The term “derivative”, as used herein, refers to the chemicalmodification of a nucleic acid encoding LGFV or the encoded LGFV.Illustrative of such modifications would be replacement of hydrogen byan alkyl, acyl, or amino group. A nucleic acid derivative would encode apolypeptide which retains essential biological characteristics of thenatural molecule.

[0037] The term “substantially purified”, as used herein, refers tonucleic or amino acid sequences that are removed from their naturalenvironment, isolated or separated, and are at least 60% free,preferably 75% free, and most preferably 90% free from other componentswith which they are naturally associated.

[0038] “Amplification” as used herein refers to the production ofadditional copies of a nucleic acid sequence and is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart (Dieffenbach, C. W. and G. S. Dveksler (1995) PCR Primer. aLaboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.).

[0039] The term “hybridization”, as used herein, refers to any processby which a strand of nucleic acid binds with a complementary strandthrough base pairing.

[0040] The term “hybridization complex”, as used herein, refers to acomplex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary G and C bases andbetween complementary A and T bases; these hydrogen bonds may be furtherstabilized by base stacking interactions. The two complementary nucleicacid sequences hydrogen bond in an antiparallel configuration. Ahybridization complex may be formed in solution (e.g., C₀t or R₀tanalysis) or between one nucleic acid sequence present in solution andanother nucleic acid sequence immobilized on a solid support (e.g.,membranes, filters, chips, pins or glass slides to which cells have beenfixed for in situ hybridization).

[0041] The terms “complementary” or “complementarity”, as used herein,refer to the natural binding of polynucleotides under permissive saltand temperature conditions by base-pairing. For example, for thesequence “A-G-T” binds to the complementary sequence “T-C-A”.Complementarity between two single-stranded molecules may be “partial”,in which only some of the nucleic acids bind, or it may be complete whentotal complementarity exists between the single stranded molecules. Thedegree of complementarity between nucleic acid strands has significanteffects on the efficiency and strength of hybridization between nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands.

[0042] The term “homology”, as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology(i.e., identity). A partially complementary sequence is one that atleast partially inhibits an identical sequence from hybridizing to atarget nucleic acid; it is referred to using the functional term“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization and the like) under conditions of low stringency. Asubstantially homologous sequence or probe will compete for and inhibitthe binding (i.e., the hybridization) of a completely homologoussequence or probe to the target sequence under conditions of lowstringency. This is not to say that conditions of low stringency aresuch that non-specific binding is permitted; low stringency conditionsrequire that the binding of two sequences to one another be a specific(i.e., selective) interaction. The absence of non-specific binding maybe tested by the use of a second target sequence which lacks even apartial degree of complementarity (e.g., less than about 30% identity);in the absence of non-specific binding, the probe will not hybridize tothe second non-complementary target sequence.

[0043] As known in the art, numerous equivalent conditions may beemployed to comprise either low or high stringency conditions. Factorssuch as the length and nature (DNA, RNA, base composition) of thesequence, nature of the target (DNA, RNA, base composition, presence insolution or immobilization, etc.), and the concentration of the saltsand other components (e.g., the presence or absence of formamide,dextran sulfate and/or polyethylene glycol) are considered and thehybridization solution may be varied to generate conditions of eitherlow or high stringency different from, but equivalent to, the abovelisted conditions.

[0044] The term “stringent conditions”, as used herein, is the“stringency” which occurs within a range from about Tm-5° C. (5° C.below the melting temperature (Tm) of the probe) to about 20° C. to 25°C. below Tm. As will be understood by those of skill in the art, thestringency of hybridization may be altered in order to identify ordetect identical or related polynucleotide sequences.

[0045] The term “antisense”, as used herein, refers to nucleotidesequences which are complementary to a specific DNA or RNA sequence. Theterm “antisense strand” is used in reference to a nucleic acid strandthat is complementary to the “sense” strand. Antisense molecules may beproduced by any method, including synthesis by ligating the gene(s) ofinterest in a reverse orientation to a viral promoter which permits thesynthesis of a complementary strand. Once introduced into a cell, thistranscribed strand combines with natural sequences produced by the cellto form duplexes. These duplexes then block either the furthertranscription or translation. In this manner, mutant phenotypes may begenerated. The designation “negative” is sometimes used in reference tothe antisense strand, and “positive” is sometimes used in reference tothe sense strand.

[0046] The term “portion”, as used herein, with regard to a protein (asin “a portion of a given protein”) refers to fragments of that protein.The fragments may range in size from four amino acid residues to theentire amino acid sequence minus one amino acid. Thus, a protein“comprising at least a portion of the amino acid sequence of SEQ IDNO:1” encompasses the full-length human LGFV and fragments thereof.

[0047] “Transformation”, as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. It may occur undernatural or artificial conditions using various methods well known in theart. Transformation may rely on any known method for the insertion offoreign nucleic acid sequences into a prokaryotic or eukaryotic hostcell. The method is selected based on the host cell being transformedand may include, but is not limited to, viral infection,electroporation, lipofection, and particle bombardment. Such“transformed” cells include stably transformed cells in which theinserted DNA is capable of replication either as an autonomouslyreplicating plasmid or as part of the host chromosome. They also includecells which transiently express the inserted DNA or RNA for limitedperiods of time.

[0048] The term “antigenic determinant”, as used herein, refers to thatportion of a molecule that makes contact with a particular antibody(i.e., an epitope). When a protein or fragment of a protein is used toimmunize a host animal, numerous regions of the protein may induce theproduction of antibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as antigenic determinants. An antigenic determinant maycompete with the intact antigen (i.e., the immunogen used to elicit theimmune response) for binding to an antibody.

[0049] The terms “specific binding” or “specifically binding”, as usedherein, in reference to the interaction of an antibody and a protein orpeptide, mean that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words, the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A”, the presence of aprotein containing epitope A (or free, unlabeled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

[0050] The term “sample”, as used herein, is used in its broadest sense.A biological sample suspected of containing nucleic acid encoding LGFVor fragments thereof may comprise a cell, chromosomes isolated from acell (e.g., a spread of metaphase chromosomes), genomic DNA (in solutionor bound to a solid support such as for Southern analysis), RNA (insolution or bound to a solid support such as for northern analysis),cDNA (in solution or bound to a solid support), an extract from cells ora tissue, and the like.

[0051] The term “correlates with expression of a polynucleotide”, asused herein, indicates that the detection of the presence of ribonucleicacid that is similar to SEQ ID NO:2 by northern analysis is indicativeof the presence of mRNA encoding LGFV in a sample and thereby correlateswith expression of the transcript from the polynucleotide encoding theprotein.

[0052] “Alterations” in the polynucleotide of SEQ ID NO:2, as usedherein, comprise any alteration in the sequence of polynucleotidesencoding LGFV including deletions, insertions, and point mutations thatmay be detected using hybridization assays. Included within thisdefinition is the detection of alterations to the genomic DNA sequencewhich encodes LGFV (e.g., by alterations in the pattern of restrictionfragment length polymorphisms capable of hybridizing to SEQ ID NO:2),the inability of a selected fragment of SEQ ID NO:2 to hybridize to asample of genomic DNA (e.g., using allele-specific oligonucleotideprobes), and improper or unexpected hybridization, such as hybridizationto a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding LGFV (e.g., using fluorescent in situhybridization [FISH] to metaphase chromosomes spreads).

[0053] As used herein, the term “antibody” refers to intact molecules aswell as fragments thereof, such as Fa, F(ab′)₂, and Fv, which arecapable of binding the epitopic determinant. Antibodies that bind LGFVpolypeptides can be prepared using intact polypeptides or fragmentscontaining small peptides of interest as the immunizing antigen. Thepolypeptide or peptide used to immunize an animal can be derived fromthe transition of RNA or synthesized chemically, and can be conjugatedto a carrier protein, if desired. Commonly used carriers that arechemically coupled to peptides include bovine serum albumin andthyroglobulin. The coupled peptide is then used to immunize the animal(e.g., a mouse, a rat, or a rabbit).

[0054] The term “humanized antibody”, as used herein, refers to antibodymolecules in which amino acids have been replaced in the non-antigenbinding regions in order to more closely resemble a human antibody,while still retaining the original binding ability.

[0055] The Invention

[0056] The invention is based on the discovery of a novel lung growthfactor variant, (LGFV), the polynucleotides encoding LGFV, and the useof these sequences in the diagnosis, prevention, and treatment ofvarious infections; pregnancy, autoimmune disorders, vascular diseases,and cancers.

[0057] Nucleic acids encoding the human LGFV of the present inventionwere first identified in Incyte Clone 876242 from the asthmatic lungcDNA library (LUNGAST01) through a computer-generated search for aminoacid sequence alignments. A consensus sequence, SEQ ID NO:2, was derivedfrom the following overlapping and/or extended nucleic acid sequences:Incyte Clones 157389 and 157561 (THP1PLB02),180392 (SINTNOT13), 269860(HNT2NOT01), 667981 (SCORNOT01), 876242 (LUNGAST01), 917859 (BRSTNOT04),938690 (CERVNOT01), 1336691 (COLNNOT11), and 1344641 (PROSNOT11 ).

[0058] In one embodiment, the invention encompasses a polypeptidecomprising the amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A,1B, and 1C. LGFV is 235 amino acids in length and lacks any potentialN-linked glycosylation sites. LGFV has chemical and structural homologywith hepatoma derived growth factor (GI 598956; SEQ ID NO:3). Inparticular, LGFV and hepatoma derived growth factor share 75% identityover the first 196 nucleotides which encode the proteins (FIGS. 3A, 3B,and 3C), and 90% identity over the entire length of the nucleotide andamino acid sequences (FIGS. 2, 3A, 3B, and 3C). As illustrated by FIGS.4A and 4B, LGFV and lung growth factor have rather similarhydrophobicity plots.

[0059] Northern analysis (FIGS. 5A, 5B, 5C, 5D, and 5E) shows theexpression of this sequence in 115 libraries, at least 51% of which areimmortalized or cancerous and at least 28% of which involve immuneresponse. Of particular note is the expression of LGFV in cancers of thebrain (˜4%), breast (˜6%) gastrointestinal tract (˜7%), lungs (˜10%),pancreas (˜3%), male reproductive system (˜9%), urinary tract (˜3%),thyroid (˜2%) and female reproductive system (˜3%) and in leukocytesincluding granulocytes, lymphocytes, and macrophages. In many of thesetissues, particularly those of the brain, breast, thyroid and those ofthe female and male reproductive systems, cell proliferation may involveassociation between LGFV and steroid hormone receptors.

[0060] The invention also encompasses LGFV variants. A preferred LGFVvariant is one having at least 90% amino acid sequence similarity to theLGFV amino acid sequence (SEQ ID NO:1). A most preferred LGFV variant isone having at least 95% amino acid sequence similarity to SEQ ID NO:1.

[0061] The invention also encompasses polynucleotides which encode LGFV.Accordingly, any nucleic acid sequence which encodes the amino acidsequence of LGFV can be used to generate recombinant molecules whichexpress LGFV. In a particular embodiment, the invention encompasses thepolynucleotide comprising the nucleic acid sequence of SEQ ID NO:2 asshown in FIGS. 1A, 1B, and 1C.

[0062] It will be appreciated by those skilled in the art that as aresult of the degeneracy of the genetic code, a multitude of nucleotidesequences encoding LGFV, some bearing minimal homology to the nucleotidesequences of any known and naturally occurring gene, may be produced.Thus, the invention contemplates each and every possible variation ofnucleotide sequence that could be made by selecting combinations basedon possible codon choices. These combinations are made in accordancewith the standard triplet genetic code as applied to the nucleotidesequence of naturally occurring LGFV, and all such variations are to beconsidered as being specifically disclosed.

[0063] Although nucleotide sequences which encode LGFV and its variantsare preferably capable of hybridizing to the nucleotide sequence of thenaturally occurring LGFV under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding LGFV or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding LGFV and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

[0064] The invention also encompasses production of DNA sequences, orportions thereof, which encode LGFV and its derivatives, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents that are well known in the art at the time of thefiling of this application. Moreover, synthetic chemistry may be used tointroduce mutations into a sequence encoding LGFV or any portionthereof.

[0065] Also encompassed by the invention are polynucleotide sequencesthat are capable of hybridizing to the claimed nucleotide sequences, andin particular, those shown in SEQ ID NO:2, under various conditions ofstringency. Hybridization conditions are based on the meltingtemperature (Tm) of the nucleic acid binding complex or probe, as taughtin Wahl, G. M. and S. L. Berger (1987; Methods Enzymol. 152:399-407) andKimmel, A. R. (1987; Methods Enzymol. 152:507-511), and may be used at adefined stringency.

[0066] Altered nucleic acid sequences encoding LGFV which areencompassed by the invention include deletions, insertions, orsubstitutions of different nucleotides resulting in a polynucleotidethat encodes the same or a functionally equivalent LGFV. The encodedprotein may also contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent LGFV. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues as long as the biological activity of LGFV is retained. Forexample, negatively charged amino acids may include aspartic acid andglutamic acid; positively charged amino acids may include lysine andarginine; and amino acids with uncharged polar head groups havingsimilar hydrophilicity values may include leucine, isoleucine, andvaline; glycine and alanine; asparagine and glutamine; serine andthreonine; phenylalanine and tyrosine.

[0067] Also included within the scope of the present invention arealleles of the genes encoding LGFV. As used herein, an “allele” or“allelic sequence” is an alternative form of the gene which may resultfrom at least one mutation in the nucleic acid sequence. Alleles mayresult in altered mRNAs or polypeptides whose structure or function mayor may not be altered. Any given gene may have none, one, or manyallelic forms. Common mutational changes which give rise to alleles aregenerally ascribed to natural deletions, additions, or substitutions ofnucleotides. Each of these types of changes may occur alone, or incombination with the others, one or more times in a given sequence.

[0068] Methods for DNA sequencing which are well known and generallyavailable in the art may be used to practice any embodiments of theinvention. The methods may employ such enzymes as the Klenow fragment ofDNA polymerase I, SEQUENASE (U.S. Biochemical Corp, Cleveland, Ohio.),Taq polymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of recombinant polymerases andproofreading exonucleases such as the ELONGASE Amplification Systemmarketed by Gibco BRL (Gaithersburg, Md). Preferably, the process isautomated with machines such as the Hamilton Micro Lab 2200 (Hamilton,Reno, Nev.), Peltier Thermal Cycler (PTC200; M. J. Research, Watertown,Mass.) and the ABI 377 DNA sequencers (Perkin Elmer).

[0069] The nucleic acid sequences encoding LGFV may be extendedutilizing a partial nucleotide sequence and employing various methodsknown in the art to detect upstream sequences such as promoters andregulatory elements. For example, one method which may be employed,“restriction-site” PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus (Sarkar, G. (1993) PCR MethodsApplic. 2:318-322). In particular, genomic DNA is first amplified in thepresence of primer to linker sequence and a primer specific to the knownregion. The amplified sequences are then subjected to a second round ofPCR with the same linker primer and another specific primer internal tothe first one. Products of each round of PCR are transcribed with anappropriate RNA polymerase and sequenced using reverse transcriptase.

[0070] Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region (Triglia, T. et al. (1988)Nucleic Acids Res. 16:8186). The primers may be designed using OLIGO4.06 Primer Analysis software (National Biosciences Inc., Plymouth,Minn.), or another appropriate program, to be 22-30 nucleotides inlength, to have a GC content of 50% or more, and to anneal to the targetsequence at temperatures about 68°-72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

[0071] Another method which may be used is capture PCR which involvesPCR amplification of DNA fragments adjacent to a known sequence in humanand yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCRMethods Applic. 1:111-119). In this method, multiple restriction enzymedigestions and ligations may also be used to place an engineereddouble-stranded sequence into an unknown portion of the DNA moleculebefore performing PCR.

[0072] Another method which may be used to retrieve unknown sequences isthat of Parker, J. D. et al. (1991; Nucleic Acids Res. 19:3055-3060).Additionally, one may use PCR, nested primers, and PROMOTERFINDERlibraries to walk in genomic DNA (Clontech, Palo Alto, Calif.). Thisprocess avoids the need to screen libraries and is useful in findingintron/exon junctions.

[0073] When screening for full-length cDNAs, it is preferable to uselibraries that have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable, in that they will contain moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into the 5′ and 3′non-transcribed regulatory regions.

[0074] Capillary electrophoresis systems which are commerciallyavailable may be used to analyze the size or confirm the nucleotidesequence of sequencing or PCR products. In particular, capillarysequencing may employ flowable polymers for electrophoretic separation,four different fluorescent dyes (one for each nucleotide) which arelaser activated, and detection of the emitted wavelengths by a chargecoupled device camera. Output/light intensity may be converted toelectrical signal using appropriate software (e.g. GENOTYPER andSEQUENCE NAVIGATOR, Perkin Elmer) and the entire process from loading ofsamples to computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

[0075] In another embodiment of the invention, polynucleotide sequencesor fragments thereof which encode LGFV, or fusion proteins or functionalequivalents thereof, may be used in recombinant DNA molecules to directexpression of LGFV in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and these sequences may be used to clone and expressLGFV.

[0076] As will be understood by those of skill in the art, it may beadvantageous to produce LGFV-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce a recombinant RNAtranscript having desirable properties, such as a half-life which islonger than that of a transcript generated from the naturally occurringsequence.

[0077] The nucleotide sequences of the present invention can beengineered using methods generally known in the art in order to alterLGFV encoding sequences for a variety of reasons, including but notlimited to, alterations which modify the cloning, processing, and/orexpression of the gene product. DNA shuffling by random fragmentationand PCR reassembly of gene fragments and synthetic oligonucleotides maybe used to engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, or introduce mutations, and so forth.

[0078] In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding LGFV may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of LGFV activity, it may be useful toencode a chimeric LGFV protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the LGFV encoding sequence and theheterologous protein sequence, so that LGFV may be cleaved and purifiedaway from the heterologous moiety.

[0079] In another embodiment, sequences encoding LGFV may besynthesized, in whole or in part, using chemical methods well known inthe art (see Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser. 225-232).Alternatively, the protein itself may be produced using chemical methodsto synthesize the amino acid sequence of LGFV, or a portion thereof. Forexample, peptide synthesis can be performed using various solid-phasetechniques (Roberge, J. Y. et al. (1995) Science 269:202-204) andautomated synthesis may be achieved, for example, using the ABI 431APeptide Synthesizer (Perkin Elmer).

[0080] The newly synthesized peptide may be substantially purified bypreparative high performance liquid chromatography (e.g., Creighton, T.(1983) Proteins, Structures and Molecular Principles, W. H. Freeman andCo., New York, N.Y.). The composition of the synthetic peptides may beconfirmed by amino acid analysis or sequencing (e.g., the Edmandegradation procedure; Creighton, supra). Additionally, the amino acidsequence of LGFV, or any part thereof, may be altered during directsynthesis and/or combined using chemical methods with sequences fromother proteins, or any part thereof, to produce a variant polypeptide.

[0081] In order to express a biologically active LGFV, the nucleotidesequences encoding LGFV or functional equivalents, may be inserted intoappropriate expression vector, i.e., a vector which contains thenecessary elements for the transcription and translation of the insertedcoding sequence.

[0082] Methods which are well known to those skilled in the art may beused to construct expression vectors containing sequences encoding LGFVand appropriate transcriptional and translational control elements.These methods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. Such techniques aredescribed in Sambrook, J. et al. (1989) Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Plainview, N.Y., and Ausubel, F. M. etal. (1989) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y.

[0083] A variety of expression vector/host systems may be utilized tocontain and express sequences encoding LGFV. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus, CaMV; tobacco mosaic virus, TMV) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems.

[0084] The “control elements” or “regulatory sequences” are thosenon-translated regions of the vector—enhancers, promoters, 5′ and 3′untranslated regions—which interact with host cellular proteins to carryout transcription and translation. Such elements may vary in theirstrength and specificity. Depending on the vector system and hostutilized, any number of suitable transcription and translation elements,including constitutive and inducible promoters, may be used. Forexample, when cloning in bacterial systems, inducible promoters such asthe hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 plasmid (Gibco BRL) and the like may be used.The baculovirus polyhedrin promoter may be used in insect cells.Promoters or enhancers derived from the genomes of plant cells (e.g.,heat shock, RUBISCO; and storage protein genes) or from plant viruses(e.g., viral promoters or leader sequences) may be cloned into thevector. In mammalian cell systems, promoters from mammalian genes orfrom mammalian viruses are preferable. If it is necessary to generate acell line that contains multiple copies of the sequence encoding LGFV,vectors based on SV40 or EBV may be used with an appropriate selectablemarker.

[0085] In bacterial systems, a number of expression vectors may beselected depending upon the use intended for LGFV. For example, whenlarge quantities of LGFV are needed for the induction of antibodies,vectors which direct high level expression of fusion proteins that arereadily purified may be used. Such vectors include, but are not limitedto, the multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding LGFV may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced; pIN vectors (Van Heeke, G. and S. M. Schuster(1989) J. Biol. Chem. 264:5503-5509); and the like. pGEX vectors(Promega, Madison, Wis.) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

[0086] In the yeast, Saccharomyces cerevisiae, a number of vectorscontaining constitutive or inducible promoters such as alpha factor,alcohol oxidase, and PGH may be used. For reviews, see Ausubel et al.(supra) and Grant et al. (1987) Methods Enzymol. 153:516-544.

[0087] In cases where plant expression vectors are used, the expressionof sequences encoding LGFV may be driven by any of a number ofpromoters. For example, viral promoters such as the 35S and 19Spromoters of CaMV may be used alone or in combination with the omegaleader sequence from TMV (Takamatsu, N. (1987) EMBO J. 6:307-311).Alternatively, plant promoters such as the small subunit of RUBISCO orheat shock promoters may be used (Coruzzi, G. et al. (1984) EMBO J.3:1671-1680; Broglie, R. et al. (1984) Science 224:838-843; and Winter,J. et al. (1991) Results Probl. Cell Differ. 17:85-105). Theseconstructs can be introduced into plant cells by direct DNAtransformation or pathogen-mediated transfection. Such techniques aredescribed in a number of generally available reviews (see, for example,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.

[0088] An insect system may also be used to express LGFV. For example,in one such system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes in Spodopterafrugiperda cells or in Trichoplusia larvae. The sequences encoding LGFVmay be cloned into a non-essential region of the virus, such as thepolyhedrin gene, and placed under control of the polyhedrin promoter.Successful insertion of LGFV will render the polyhedrin gene inactiveand produce recombinant virus lacking coat protein. The recombinantviruses may then be used to infect, for example, S. frugiperda cells orTrichoplusia larvae in which LGFV may be expressed (Engelhard, E. K. etal. (1994) Proc. Nat. Acad. Sci. 91:3224-3227).

[0089] In mammalian host cells, a number of viral-based expressionsystems may be utilized. In cases where an adenovirus is used as anexpression vector, sequences encoding LGFV may be ligated into anadenovirus transcription/translation complex consisting of the latepromoter and tripartite leader sequence. Insertion in a non-essential E1or E3 region of the viral genome may be used to obtain a viable viruswhich is capable of expressing LGFV in infected host cells (Logan, J.and Shenk, T. (1984) Proc. Natl. Acad. Sci. 81:3655-3659). In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

[0090] Specific initiation signals may also be used to achieve moreefficient translation of sequences encoding LGFV. Such signals includethe ATG initiation codon and adjacent sequences. In cases wheresequences encoding LGFV, its initiation codon, and upstream sequencesare inserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a portion thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers which are appropriate for theparticular cell system which is used, such as those described in theliterature (Scharf, D. et al. (1994) Results Probl. Cell Differ.20:125-162).

[0091] In addition, a host cell strain may be chosen for its ability tomodulate the expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding and/orfunction. Different host cells such as CHO, HeLa, MDCK, HEK293, andWI38, which have specific cellular machinery and characteristicmechanisms for such post-translational activities, may be chosen toensure the correct modification and processing of the foreign protein.

[0092] For long-term, high-yield production of recombinant proteins,stable expression is preferred. For example, cell lines which stablyexpress LGFV may be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for 1-2 days in an enriched media before they are switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

[0093] Any number of selection systems may be used to recovertransformed cell lines.

[0094] These include, but are not limited to, the herpes simplex virusthymidine kinase (Wigler, M. et al. (1977) Cell 11:223-32) and adeninephosphoribosyltransferase (Lowy, I. et al. (1980) Cell 22:817-23) geneswhich can be employed in tk⁻ or aprt⁻ cells, respectively. Also,antimetabolite, antibiotic or herbicide resistance can be used as thebasis for selection; for example, dhfr which confers resistance tomethotrexate (Wigler, M. et al. (1980) Proc. Natl. Acad. Sci.77:3567-70); npt, which confers resistance to the aminoglycosidesneomycin and G-418 (Colbere-Garapin, F. et al (1981) J. Mol. Biol.150:1-14) and als or pat, which confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively (Murry, supra).Additional selectable genes have been described, for example, trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine (Hartman,S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-51).Recently, the use of visible markers has gained popularity with suchmarkers as anthocyanins, β glucuronidase and its substrate GUS, andluciferase and its substrate luciferin, being widely used not only toidentify transformants, but also to quantify the amount of transient orstable protein expression attributable to a specific vector system(Rhodes, C. A. et al. (1995) Methods Mol. Biol. 55:121-131).

[0095] Although the presence/absence of marker gene expression suggeststhat the gene of interest is also present, its presence and expressionmay need to be confirmed. For example, if the sequence encoding LGFV isinserted within a marker gene sequence, recombinant cells containingsequences encoding LGFV can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding LGFV under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

[0096] Alternatively, host cells which contain the nucleic acid sequenceencoding LGFV and express LGFV may be identified by a variety ofprocedures known to those of skill in the art.

[0097] These procedures include, but are not limited to, DNA-DNA orDNA-RNA hybridizations and protein bioassay or immunoassay techniqueswhich include membrane, solution, or chip based technologies for thedetection and/or quantification of nucleic acid or protein.

[0098] The presence of polynucleotide sequences encoding LGFV can bedetected by DNA-DNA or DNA-RNA hybridization or amplification usingprobes or portions or fragments of polynucleotides encoding LGFV.Nucleic acid amplification based assays involve the use ofoligonucleotides or oligomers based on the sequences encoding LGFV todetect transformants containing DNA or RNA encoding LGFV. As used herein“oligonucleotides” or “oligomers” refer to a nucleic acid sequence of atleast about 10 nucleotides and as many as about 60 nucleotides,preferably about 15 to 30 nucleotides, and more preferably about 20-25nucleotides, which can be used as a probe or amplimer.

[0099] A variety of protocols for detecting and measuring the expressionof LGFV, using either polyclonal or monoclonal antibodies specific forthe protein are known in the art. Examples include enzyme-linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescenceactivated cell sorting (FACS). A two-site, monoclonal-based immunoassayutilizing monoclonal antibodies reactive to two non-interfering epitopeson LGFV is preferred, but a competitive binding assay may be employed.These and other assays are described, among other places, in Hampton, R.et al. (1990; Serological Methods, a Laboratory Manual, APS Press, StPaul, Minn.) and Maddox, D. E. et al. (1983; J. Exp. Med.158:1211-1216).

[0100] A wide variety of labels and conjugation techniques are known bythose skilled in the art and may be used in various nucleic acid andamino acid assays. Means for producing labeled hybridization or PCRprobes for detecting sequences related to polynucleotides encoding LGFVinclude oligolabeling, nick translation, end-labeling or PCRamplification using a labeled nucleotide. Alternatively, the sequencesencoding LGFV, or any portions thereof may be cloned into a vector forthe production of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits (Pharmacia & Upjohn, (Kalamazoo,Mich.); Promega (Madison Wis.); and U.S. Biochemical Corp. (Cleveland,Ohio.)). Suitable reporter molecules or labels, which may be used,include radionuclides, enzymes, fluorescent, chemiluminescent, orchromogenic agents as well as substrates, cofactors, inhibitors,magnetic particles, and the like.

[0101] Host cells transformed with nucleotide sequences encoding LGFVmay be cultured under conditions suitable for the expression andrecovery of the protein from cell culture.

[0102] The protein produced by a recombinant cell may be secreted orcontained intracellularly depending on the sequence and/or the vectorused. As will be understood by those of skill in the art, expressionvectors containing polynucleotides which encode LGFV may be designed tocontain signal sequences which direct secretion of LGFV through aprokaryotic or eukaryotic cell membrane. Other recombinant constructionsmay be used to join sequences encoding LGFV to nucleotide sequenceencoding a polypeptide domain which will facilitate purification ofsoluble proteins. Such purification facilitating domains include, butare not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.) between the purificationdomain and LGFV may be used to facilitate purification. One suchexpression vector provides for expression of a fusion protein containingLGFV and a nucleic acid encoding 6 histidine residues preceding athioredoxin or an enterokinase cleavage site. The histidine residuesfacilitate purification on IMAC (immobilized metal ion affinitychromatography as described in Porath, J. et al. (1992, Prot. Exp.Purif. 3:263-281) while the enterokinase cleavage site provides a meansfor purifying LGFV from the fusion protein. A discussion of vectorswhich contain fusion proteins is provided in Kroll, D. J. et al. (1993;DNA Cell Biol. 12:441-453).

[0103] In addition to recombinant production, fragments of LGFV may beproduced by direct peptide synthesis using solid-phase techniquesMerrifield J. (1963) J. Am. Chem. Soc. 85:2149-2154). Protein synthesismay be performed using manual techniques or by automation. Automatedsynthesis may be achieved, for example, using Applied Biosystems 431APeptide Synthesizer (Perkin Elmer). Various fragments of LGFV may bechemically synthesized separately and combined using chemical methods toproduce the full length molecule

[0104] Therapeutics

[0105] Based on the chemical and structural homology among LGFV (SEQ IDNO:1) and HDGFs from man (SEQ ID NO:3) and mouse (SEQ ID NO:4), theexpression of LGFV in brain, breast, female reproductive system, fetaltissues, gastrointestinal tract, lungs, pancreas, male reproductivesystem, thyroid and urinary tract, in leukocytes including granulocytes,lymphocytes, and macrophages, and in many immortalized cell lines, LGFVappears to play a role in growth and development as well as ininfections, spermatogenesis, autoimmune diseases, vascular conditionsand cancers.

[0106] Therefore, in one embodiment, LGFV or a fragment or derivativethereof may be used to treat cells in vivo or ex vivo for the purposesof tissue or organ regeneration. This embodiment would be of particularbenefit in the proliferation and differentiation of bone marrow, nerve,pancreatic or renal cells.

[0107] In another embodiment, a vector capable of expressing LGFV, or afragment or derivative thereof, may also be administered to a cellculture or a subject for ex vivo or in vivo therapy as previously above.

[0108] In one aspect, antibodies which are specific for LGFV may be useddirectly as an antagonist or indirectly as a targeting or deliverymechanism for bringing a pharmaceutical agent to cells or tissues whichexpress excessive amounts of LGFV.

[0109] In one embodiment, antagonists or inhibitors of LGFV may beadministered to a subject to prevent cancerous cell proliferation and/ortissue damage due to excessive leukocytes produced during autoimmune orimmunological responses. Such cancers may include, but are not limitedto, leukemia, lymphomas or carcinomas. Excessive immunological responsemay be that which is attributed to, but is not limited to, viral (AIDS),bacterial (pulmonary pneumonia, hepatitis or septic shock), fungal(histoplasmosis, leprosy) or helminthic/parasitic infections; allergiesor asthma; arteriosclerosis, atherogenesis or collagen vasculardiseases; and autoimmune diseases such as hemolytic anemia, biliarycirrhosis, Crohn's disease, diabetes mellitus, lupus erythematosus,multiple sclerosis, myasthenia gravis, or rheumatoid arthritis.

[0110] In another aspect, inhibitors of LGFV can be administered in asuitable formulation to prevent spermatogenesis in mammalianreproductive tissues, thereby effecting birth control.

[0111] In another embodiment, a vector expressing antisense of thepolynucleotide encoding LGFV may be administered to a subject to preventgrowth and development of cancerous cells or to modulate the progressionof the autoimmune diseases listed above or the proliferation ofleukocytes which cause tissue destruction and are associated withinfections or immunological response as described above.

[0112] In other embodiments, any of the therapeutic proteins,antagonists, antibodies, agonists, antisense sequences or vectorsdescribed above may be administered in combination with otherappropriate therapeutic agents. Selection of the appropriate agents foruse in combination therapy may be made by one of ordinary skill in theart, according to conventional pharmaceutical principles. Thecombination of therapeutic agents may act synergistically to effect thetreatment or prevention of the various disorders described above. Usingthis approach, one may be able to achieve therapeutic efficacy withlower dosages of each agent, thus reducing the potential for adverseside effects.

[0113] Antagonists or inhibitors of LGFV may be produced using methodswhich are generally known in the art. In particular, purified LGFV maybe used to produce antibodies or to screen libraries of pharmaceuticalagents to identify those which specifically bind LGFV.

[0114] Antibodies which are specific for LGFV may be used directly as anantagonist, or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express LGFV.The antibodies may be generated using methods that are well known in theart. Such antibodies may include, but are not limited to, polyclonal,monoclonal, chimeric, single chain, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies, (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

[0115] For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others, may be immunized by injectionwith LGFV or any fragment or oligopeptide thereof which has immunogenicproperties. Depending on the host species, various adjuvants may be usedto increase immunological response. Such adjuvants include, but are notlimited to, Freund's, mineral gels such as aluminum hydroxide, andsurface active substances such as lysolecithin, pluronic polyols,polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, anddinitrophenol. Among adjuvants used in humans, BCG (bacilliCalmette-Guerin) and Corynebacterium parvum are especially preferable.

[0116] It is preferred that the peptides, fragments, or oligopeptidesused to induce antibodies to LGFV have an amino acid sequence consistingof at least five amino acids, and more preferably at least 10 aminoacids. It is also preferable that they are identical to a portion of theamino acid sequence of the natural protein, and they may contain theentire amino acid sequence of a small, naturally occurring molecule.Short stretches of LGFV amino acids may be fused with those of anotherprotein such as keyhole limpet hemocyanin and antibody produced againstthe chimeric molecule.

[0117] Monoclonal antibodies to LGFV may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120).

[0118] In addition, techniques developed for the production of “chimericantibodies”, the splicing of mouse antibody genes to human antibodygenes to obtain a molecule with appropriate antigen specificity andbiological activity can be used (Morrison, S. L. et al. (1984) Proc.Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al. (1984) Nature312:604-608; Takeda, S. et al. (1985) Nature 314:452-454).Alternatively, techniques described for the production of single chainantibodies may be adapted, using methods known in the art, to produceLGFV-specific single chain antibodies. Antibodies with relatedspecificity, but of distinct idiotypic composition, may be generated bychain shuffling from random combinatorial immunoglobulin libraries(Kang, A. S. et al. (1991) Proc. Natl. Acad. Sci. 88:11120-3).

[0119] Antibodies may also be produced by inducing in vivo production inthe lymphocyte population or by screening recombinant immunoglobulinlibraries or panels of highly specific binding reagents as disclosed inthe literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; Winter, G. et al. (1991) Nature 349:293-299).

[0120] Antibody fragments which contain specific binding sites for LGFVmay also be generated. For example, such fragments include, but are notlimited to, the F(ab′)2 fragments which can be produced by pepsindigestion of the antibody molecule and the Fab fragments which can begenerated by reducing the disulfide bridges of the F(ab′)2 fragments.Alternatively, Fab expression libraries may be constructed to allowrapid and easy identification of monoclonal Fab fragments with thedesired specificity (Huse, W. D. et al. (1989) Science 254:1275-1281).

[0121] Various immunoassays may be used for screening to identifyantibodies having the desired specificity. Numerous protocols forcompetitive binding or immunoradiometric assays using either polyclonalor monoclonal antibodies with established specificities are well knownin the art. Such immunoassays typically involve the measurement ofcomplex formation between LGFV and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering LGFV epitopes is preferred, but a competitivebinding assay may also be employed (Maddox, supra).

[0122] In another embodiment of the invention, the polynucleotidesencoding LGFV, or any fragment thereof, or antisense molecules, may beused for therapeutic purposes. In one aspect, antisense to thepolynucleotide encoding LGFV may be used in situations in which it wouldbe desirable to block the transcription of the mRNA. In particular,cells may be transformed with sequences complementary to polynucleotidesencoding LGFV. Thus, antisense molecules may be used to modulate LGFVactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligomers or largerfragments, can be designed from various locations along the coding orcontrol regions of sequences encoding LGFV.

[0123] Expression vectors derived from retroviruses, adenovirus, herpesor vaccinia viruses, or from various bacterial plasmids may be used fordelivery of nucleotide sequences to the targeted organ, tissue or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct recombinant vectors which will express antisensemolecules complementary to the polynucleotides of the gene encodingLGFV. These techniques are described both in Sambrook et al. (supra) andin Ausubel et al. (supra).

[0124] Genes encoding LGFV can be turned off by transforming a cell ortissue with expression vectors which express high levels of apolynucleotide or fragment thereof which encodes LGFV. Such constructsmay be used to introduce untranslatable sense or antisense sequencesinto a cell. Even in the absence of integration into the DNA, suchvectors may continue to transcribe RNA molecules until they are disabledby endogenous nucleases. Transient expression may last for a month ormore with a non-replicating vector and even longer if appropriatereplication elements are part of the vector system.

[0125] As mentioned above, modifications of gene expression can beobtained by designing antisense molecules, DNA, RNA, or PNA, to thecontrol regions of the gene encoding LGFV, i.e., the promoters,enhancers, and introns. Oligonucleotides derived from the transcriptioninitiation site, e.g., between positions −10 and +10 from the startsite, are preferred. Similarly, inhibition can be achieved using “triplehelix” base-pairing methodology. Triple helix pairing is useful becauseit causes inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature (Gee, J. E. et al. (1994) In:

[0126] Huber, B. E. and B. I. Carr, Molecular and ImmunologicApproaches, Futura Publishing Co., Mt. Kisco, N.Y.). The antisensemolecules may also be designed to block translation of mRNA bypreventing the transcript from binding to ribosomes.

[0127] Ribozymes, enzymatic RNA molecules, may also be used to catalyzethe specific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Exampleswhich may be used include engineered hammerhead motif ribozyme moleculesthat can specifically and efficiently catalyze endonucleolytic cleavageof sequences encoding LGFV.

[0128] Specific ribozyme cleavage sites within any potential RNA targetare initially identified by scanning the target molecule for ribozymecleavage sites which include the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides corresponding to the region of the target genecontaining the cleavage site may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

[0129] Antisense molecules and ribozymes of the invention may beprepared by any method known in the art for the synthesis of nucleicacid molecules. These include techniques for chemically synthesizingoligonucleotides such as solid phase phosphoramidite chemical synthesis.Alternatively, RNA molecules may be generated by in vitro and in vivotranscription of DNA sequences encoding LGFV. Such DNA sequences may beincorporated into a wide variety of vectors with suitable RNA polymerasepromoters such as T7 or SP6. Alternatively, these cDNA constructs thatsynthesize antisense RNA constitutively or inducibly can be introducedinto cell lines, cells, or tissues.

[0130] RNA molecules may be modified to increase intracellular stabilityand half-life. Possible modifications include, but are not limited to,the addition of flanking sequences at the 5′ and/or 3′ ends of themolecule or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

[0131] Many methods for introducing vectors into cells or tissues areavailable and equally suitable for use in vivo, in vitro, and ex vivo.For ex vivo therapy, vectors may be introduced into stem cells takenfrom the patient and clonally propagated for autologous transplant backinto that same patient. Delivery by transfection and by liposomeinjections may be achieved using methods which are well known in theart.

[0132] Any of the therapeutic methods described above may be applied toany subject in need of such therapy, including, for example, mammalssuch as dogs, cats, cows, horses, rabbits, monkeys, and most preferably,humans.

[0133] An additional embodiment of the invention relates to theadministration of a pharmaceutical composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of LGFV,antibodies to LGFV, mimetics, agonists, antagonists, or inhibitors ofLGFV. The compositions may be administered alone or in combination withat least one other agent, such as stabilizing compound, which may beadministered in any sterile, biocompatible pharmaceutical carrier,including, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs or hormones.

[0134] The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

[0135] In addition to the active ingredients, these pharmaceuticalcompositions may contain suitable pharmaceutically-acceptable carrierscomprising excipients and auxiliaries which facilitate processing of theactive compounds into preparations which can be used pharmaceutically.Further details on techniques for formulation and administration may befound in the latest edition of Remington's Pharmaceutical Sciences(Maack Publishing Co., Easton, Pa.).

[0136] Pharmaceutical compositions for oral administration can beformulated using pharmaceutically acceptable carriers well known in theart in dosages suitable for oral administration. Such carriers enablethe pharmaceutical compositions to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions, and thelike, for ingestion by the patient.

[0137] Pharmaceutical preparations for oral use can be obtained throughcombination of active compounds with solid excipient, optionallygrinding a resulting mixture, and processing the mixture of granules,after adding suitable auxiliaries, if desired, to obtain tablets ordragee cores. Suitable excipients are carbohydrate or protein fillers,such as sugars, including lactose, sucrose, mannitol, or sorbitol;starch from corn, wheat, rice, potato, or other plants; cellulose, suchas methyl cellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums including arabic and tragacanth; andproteins such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, alginic acid, or a salt thereof, such as sodiumalginate.

[0138] Dragee cores may be used in conjunction with suitable coatings,such as concentrated sugar solutions, which may also contain gum arabic,talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/ortitanium dioxide, lacquer solutions, and suitable organic solvents orsolvent mixtures. Dyestuffs or pigments may be added to the tablets ordragee coatings for product identification or to characterize thequantity of active compound, i.e., dosage.

[0139] Pharmaceutical preparations which can be used orally includepush-fit capsules made of gelatin, as well as soft, sealed capsules madeof gelatin and a coating, such as glycerol or sorbitol. Push-fitcapsules can contain active ingredients mixed with a filler or binders,such as lactose or starches, lubricants, such as talc or magnesiumstearate, and, optionally, stabilizers. In soft capsules, the activecompounds may be dissolved or suspended in suitable liquids, such asfatty oils, liquid, or liquid polyethylene glycol with or withoutstabilizers.

[0140] Pharmaceutical formulations suitable for parenteraladministration may be formulated in aqueous solutions, preferably inphysiologically compatible buffers such as Hanks' solution, Ringer'ssolution, or physiologically buffered saline. Aqueous injectionsuspensions may contain substances which increase the viscosity of thesuspension, such as sodium carboxymethyl cellulose, sorbitol, ordextran. Additionally, suspensions of the active compounds may beprepared as appropriate oily injection suspensions. Suitable lipophilicsolvents or vehicles include fatty oils such as sesame oil, or syntheticfatty acid esters, such as ethyl oleate or triglycerides, or liposomes.Optionally, the suspension may also contain suitable stabilizers oragents which increase the solubility of the compounds to allow for thepreparation of highly concentrated solutions.

[0141] For topical or nasal administration, penetrants appropriate tothe particular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

[0142] The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

[0143] The pharmaceutical composition may be provided as a salt and canbe formed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend tobe more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1-50 mM histidine, 0.1%-2% sucrose, and 2-7% mannitol, at apH range of 4.5 to 5.5, that is combined with buffer prior to use.

[0144] After pharmaceutical compositions have been prepared, they can beplaced in an appropriate container and labeled for treatment of anindicated condition. For administration of LGFV, such labeling wouldinclude amount, frequency, and method of administration.

[0145] Pharmaceutical compositions suitable for use in the inventioninclude compositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

[0146] For any compound, the therapeutically effective dose can beestimated initially either in cell culture assays, e.g., of neoplasticcells, or in animal models, usually mice, rabbits, dogs, or pigs. Theanimal model may also be used to determine the appropriate concentrationrange and route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

[0147] A therapeutically effective dose refers to that amount of activeingredient, for example LGFV or fragments thereof, antibodies of LGFV,agonists, antagonists or inhibitors of LGFV, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., ED50 (the dose therapeutically effective in50% of the population) and LD50 (the dose lethal to 50% of thepopulation). The dose ratio of toxic to therapeutic effects is thetherapeutic index, which can be expressed as the LD₅₀/ED₅₀ ratio.Pharmaceutical compositions which exhibit large therapeutic indices arepreferred. The data obtained from cell culture assays and animal studiesis used in formulating a range of dosage for human use. The dosagecontained in such compositions is preferably within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage varies within this range depending upon the dosageform employed, sensitivity of the patient, and the route ofadministration.

[0148] The exact dosage will be determined by the practitioner, in lightof factors related to the subject that requires treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, general healthof the subject, age, weight, and gender of the subject, diet, time andfrequency of administration, drug combination(s), reactionsensitivities, and tolerance/response to therapy. Long-actingpharmaceutical compositions may be administered every 3 to 4 days, everyweek, or once every two weeks depending on half-life and clearance rateof the particular formulation.

[0149] Normal dosage amounts may vary from 0.1 to 100,000 micrograms, upto a total dose of about 1 g, depending upon the route ofadministration. Guidance as to particular dosages and methods ofdelivery is provided in the literature and generally available topractitioners in the art. Those skilled in the art will employ differentformulations for nucleotides than for proteins or their inhibitors.Similarly, delivery of polynucleotides or polypeptides will be specificto particular cells, conditions, locations, etc.

[0150] Diagnostics

[0151] In another embodiment, antibodies which specifically bind LGFVmay be used for the diagnosis of conditions or diseases characterized byexpression of LGFV, or in assays to monitor patients being treated withLGFV, agonists, antagonists or inhibitors. The antibodies useful fordiagnostic purposes may be prepared in the same manner as thosedescribed above for therapeutics. Diagnostic assays for LGFV includemethods which utilize the antibody and a label to detect LGFV in humanbody fluids or extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by joining them, eithercovalently or non-covalently, with a reporter molecule. A wide varietyof reporter molecules which are known in the art may be used, several ofwhich are described above.

[0152] A variety of protocols including ELISA, RIA, and FACS formeasuring LGFV are known in the art and provide a basis for diagnosingaltered or abnormal levels of LGFV expression. Normal or standard valuesfor LGFV expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to LGFV under conditions suitable for complex formation Theamount of standard complex formation may be quantified by variousmethods, but preferably by photometric, means. Quantities of LGFVexpressed in subject, control and disease, samples from biopsied tissuesare compared with the standard values. Deviation between standard andsubject values establishes the parameters for diagnosing disease.

[0153] In another embodiment of the invention, the polynucleotidesencoding LGFV may be used for diagnostic purposes. The polynucleotideswhich may be used include oligonucleotide sequences, antisense RNA andDNA molecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofLGFV may be correlated with disease. The diagnostic assay may be used todistinguish between absence, presence, and excess expression of LGFV,and to monitor regulation of LGFV levels during therapeuticintervention.

[0154] In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding LGFV or closely related molecules, may be used to identifynucleic acid sequences which encode LGFV. The specificity of the probe,whether it is made from a highly specific region, e.g., 10 uniquenucleotides in the 5′ regulatory region, or a less specific region,e.g., especially in the 3′ coding region, and the stringency of thehybridization or amplification (maximal, high, intermediate, or low)will determine whether the probe identifies only naturally occurringsequences encoding LGFV, alleles, or related sequences.

[0155] Probes may also be used for the detection of related sequences,and should preferably contain at least 50% of the nucleotides from anyof the LGFV encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and derived from the nucleotide sequence ofSEQ ID NO:2 or from genomic sequence including promoter, enhancerelements, and introns of the naturally occurring LGFV.

[0156] Means for producing specific hybridization probes for DNAsencoding LGFV include the cloning of nucleic acid sequences encodingLGFV or LGFV derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, commercially available, and may beused to synthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, radionuclides such as ³²P or ³⁵S, or enzymatic labels, such asalkaline phosphatase coupled to the probe via avidin/biotin couplingsystems, and the like.

[0157] Polynucleotide sequences encoding LGFV may be used for thediagnosis of conditions or diseases which are associated with excessiveexpression of LGFV. Examples include infections, pregnancy, autoimmunediseases, vascular conditions and cancers. These specifically include,but are not limited to, conditions with similar biochemical, mitogenic,or immunological properties such as viral (AIDS), bacterial (pulmonarypneumonia, hepatitis or septic shock), fungal (histoplasmosis, leprosy)or helminthic/parasitic infections; allergies or asthma; mechanicalinjury through exposure (to asbestos, coal dust, etc) or trauma;arteriosclerosis, atherogenesis or collagen vascular diseases;hereditary diseases such as autoimmune hemolytic anemia, biliarycirrhosis, Crohn's disease, diabetes mellitus, lupus erythematosus,multiple sclerosis, myasthenia gravis, or rheumatoid arthritis;spermatogenesis, and leukemia, lymphomas or carcinomas Thepolynucleotide sequences encoding LGFV may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; or in dip stick, pin, ELISA or chip assays utilizingfluids or tissues from patient biopsies to detect altered LGFVexpression. Such qualitative or quantitative methods are well known inthe art.

[0158] In a particular aspect, the nucleotide sequences encoding LGFVmay be useful in assays that detect activation or induction of variouscancers, particularly those mentioned above. The nucleotide sequencesencoding LGFV may be labeled by standard methods, and added to a fluidor tissue sample from a patient under conditions suitable for theformation of hybridization complexes. After a suitable incubationperiod, the sample is washed and the signal is quantitated and comparedwith a standard value. If the amount of signal in the biopsied orextracted sample is significantly altered from that of a comparablecontrol sample, the nucleotide sequences have hybridized with nucleotidesequences in the sample, and the presence of altered levels ofnucleotide sequences encoding LGFV in the sample indicates the presenceof the associated disease. Such assays may also be used to evaluate theefficacy of a particular therapeutic treatment regimen in animalstudies, in clinical trials, or in monitoring the treatment of anindividual patient.

[0159] In order to provide a basis for the diagnosis of diseaseassociated with expression of LGFV, a normal or standard profile forexpression is established. This may be accomplished by combining bodyfluids or cell extracts taken from normal subjects, either animal orhuman, with a sequence, or a fragment thereof, which encodes LGFV, underconditions suitable for hybridization or amplification. Standardhybridization may be quantified by comparing the values obtained fromnormal subjects with those from an experiment where a known amount of asubstantially purified polynucleotide is used. Standard values obtainedfrom normal samples may be compared with values obtained from samplesfrom patients who are symptomatic for disease. Deviation betweenstandard and subject values is used to establish the presence ofdisease.

[0160] Once disease is established and a treatment protocol isinitiated, hybridization assays may be repeated on a regular basis toevaluate whether the level of expression in the patient begins toapproximate that which is observed in the normal patient. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

[0161] With respect to cancer, the presence of a relatively high amountof transcript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

[0162] Additional diagnostic uses for oligonucleotides designed from thesequences encoding LGFV may involve the use of PCR. Such oligomers maybe chemically synthesized, generated enzymatically, or produced from arecombinant source. Oligomers will preferably consist of two nucleotidesequences, one with sense orientation (5′->3′) and another withantisense (3′<-5′), employed under optimized conditions foridentification of a specific gene or condition. The same two oligomers,nested sets of oligomers, or even a degenerate pool of oligomers may beemployed under less stringent conditions for detection and/orquantitation of closely related DNA or RNA sequences.

[0163] Methods which may also be used to quantitate the expression ofLGFV include radiolabeling or biotinylating nucleotides, coamplificationof a control nucleic acid, and standard curves onto which theexperimental results are interpolated (Melby, P. C. et al. (1993) J.Immunol. Methods, 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem.229-236). The speed of quantitation of multiple samples may beaccelerated by running the assay in an ELISA format where the oligomerof interest is presented in various dilutions and a spectrophotometricor colorimetric response gives rapid quantitation.

[0164] In another embodiment of the invention, the nucleic acidsequences which encode LGFV may also be used to generate hybridizationprobes which are useful for mapping the naturally occurring genomicsequence. The sequences may be mapped to a particular-chromosome or to aspecific region of the chromosome using well known techniques . Suchtechniques include FISH, PACS, or artificial chromosome constructions,such as yeast artificial chromosomes, bacterial artificial chromosomes,bacterial PI constructions or single chromosome cDNA libraries asreviewed in Price, C. M. (1993) Blood Rev. 7:127-134, and Trask, B. J.(1991) Trends Genet. 7:149-154.

[0165] FISH (as described in Verma et al. (1988) Human Chromosomes: AManual of Basic Techniques, Pergamon Press, New York, N.Y.) may becorrelated with other physical chromosome mapping techniques and geneticmap data. Examples of genetic map data can be found in the 1994 GenomeIssue of Science (265:1981f). Correlation between the location of thegene encoding LGFV on a physical chromosomal map and a specific disease,or predisposition to a specific disease, may help delimit the region ofDNA associated with that genetic disease. The nucleotide sequences ofthe subject invention may be used to detect differences in genesequences between normal, carrier, or affected individuals.

[0166] In situ hybridization of chromosomal preparations and physicalmapping techniques such as linkage analysis using establishedchromosomal markers may be used for extending genetic maps. Often theplacement of a gene on the chromosome of another mammalian species, suchas mouse, may reveal associated markers even if the number or arm of aparticular human chromosome is not known. New sequences can be assignedto chromosomal arms, or parts thereof, by physical mapping. Thisprovides valuable information to investigators searching for diseasegenes using positional cloning or other gene discovery techniques. Oncethe disease or syndrome has been crudely localized by genetic linkage toa particular genomic region, for example, AT to 11q22-23 (Gatti, R. A.et al. (1988) Nature 336:577-580), any sequences mapping to that areamay represent associated or regulatory genes for further investigation.The nucleotide sequence of the subject invention may also be used todetect differences in the chromosomal location due to translocation,inversion, etc. among normal, carrier, or affected individuals.

[0167] In another embodiment of the invention, LGFV, its catalytic orimmunogenic fragments or oligopeptides thereof, can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes, betweenLGFV and the agent being tested, may be measured.

[0168] Another technique for drug screening which may be used providesfor high throughput screening of compounds having suitable bindingaffinity to the protein of interest as described in published PCTapplication WO84/03564. In this method, as applied to LGFV large numbersof different small test compounds are synthesized on a solid substrate,such as plastic pins or some other surface. The test compounds arereacted with LGFV, or fragments thereof, and washed. Bound LGFV is thendetected by methods well known in the art. Purified LGFV can also becoated directly onto plates for use in the aforementioned drug screeningtechniques. Alternatively, non-neutralizing antibodies can be used tocapture the peptide and immobilize it on a solid support.

[0169] In another embodiment, one may use competitive drug screeningassays in which neutralizing antibodies capable of binding LGFVspecifically compete with a test compound for binding LGFV. In thismanner, the antibodies can be used to detect the presence of any peptidewhich shares one or more antigenic determinants with LGFV.

[0170] In additional embodiments, the nucleotide sequences which encodeLGFV may be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

[0171] The examples below are provided to illustrate the subjectinvention and are not included for the purpose of limiting theinvention.

EXAMPLES

[0172] I LUNGAST01 cDNA Library Construction

[0173] The LUNGAST01 cDNA library was constructed from cryopreservedlung purchased from Clonetics (San Diego Calif.; catalog #CC-2501,tissue lot no. 2199;). The tissue donor was a 30 year old Afro-Americanfemale who underwent elective breast reduction surgery. At the time ofsurgery, the donor was taking ferrous sulfate in preparation for thesurgery, and a routine blood test was unremarkable except for a slightelevation of serum alanine transferase. The patient reported regulartobacco use, but no associated symptoms and no prior surgery. Lungtissue was isolated from the resected tissue and allowed to proliferatebefore cryopreservation.

[0174] The cells were lysed using a Brinkmann Homogenizer PolytronPT-3000 (Brinkmann Instruments, Westbury N.J.) in guanidiniumisothiocyanate. The lysate was centrifuged over a 5.7 M CsC1 cushionusing a Beckman SW28 rotor in a Beckman L8-70M Ultracentrifuge (BeckmanInstruments) for 18 hours at 25,000 rpm at ambient temperature. The RNAwas extracted with phenol chloroform pH 8.0, precipitated using 0.3 Msodium acetate and 2.5 volumes of ethanol, resuspended in RNAse-freewater and DNase treated at 37° C. The RNA was reextracted with phenolchloroform and precipitated using sodium acetate and ethanol as before.The mRNA was then isolated using the Qiagen OLIGOTEX kit (QIAGEN Inc;

[0175] Chatsworth Calif.) and used to construct the cDNA library.

[0176] The mRNA was handled according to the recommended protocols inthe SUPERSCRIPT Plasmid System for cDNA Synthesis and Plasmid Cloning(Cat. #18248-013; Gibco/BRL). cDNAs were fractionated on a SepharoseCL4B column (Cat. #275105; Pharmacia), and those cDNAs exceeding 400 bpwere ligated into PSPORT1. The plasmid PSPORT1 was subsequentlytransformed into DH5 αcompetent cells (Cat. #18258-012; Gibco/BRL).

[0177] II Isolation and Sequencing of cDNA Clones

[0178] Plasmid DNA was released from the cells and purified using theREAL Prep 96 Plasmid kit (Catalog #26173; Qiagen, Inc). This kit enablesthe simultaneous purification of 96 samples in a 96-well format usingmulti-channel reagent dispensers. The recommended protocol was employedexcept for the following changes:1) the bacteria were cultured in 1 mlof sterile Terrific Broth (Catalog #22711, LIFE TECHNOLOGIES™,Gaithersburg, Md.) with carbenicillin at 25 mg/L and glycerol at 0.4%;2) the cultures were incubated for 19 hours and then lysed with 0.3 mlof lysis buffer; 3) following isopropanol precipitation, the plasmid DNApellet was resuspended in 0.1 ml of distilled water. After the last stepin the protocol, samples were transferred to a Beckman 96-well block forstorage at 4° C.

[0179] The cDNAs were sequenced by the method of Sanger F and A RCoulson (1975; J Mol Biol 94:441f), using a Hamilton Micro Lab 2200(Hamilton, Reno Nev.) in combination with four Peltier Thermal Cyclers(PTC200 from MJ Research, Watertown Mass.) and Applied Biosystems 377 or373 DNA Sequencing Systems; and the reading frame was determined.

[0180] III Homology Searching of cDNA Clones and Their Deduced Proteins

[0181] The nucleotide sequences (or amino acid sequences deduced fromthem) were used as query sequences against databases such as GenBank,SwissProt, BLOCKS, and Pima II. These databases which contain previouslyidentified and annotated sequences were searched for regions of homology(similarity) using BLAST, which stands for Basic Local Alignment SearchTool (Altschul (1993) supra, Altschul (1990) supra).

[0182] BLAST produces alignments of both nucleotide and amino acidsequences to determine sequence similarity. Because of the local natureof the alignments, BLAST is especially useful in determining exactmatches or in identifying homologs which may be of prokaryotic(bacterial) or eukaryotic (animal, fungal or plant) origin. Otheralgorithms such as the one described in Smith R F and T F Smith (1992Protein Engineering 5:35-51), incorporated herein by reference, can beused when dealing with primary sequence patterns and secondary structuregap penalties. As disclosed in this application, the sequences havelengths of at least 49 nucleotides, and no more than 12% uncalled bases(where N is recorded rather than A, C., G, or T).

[0183] The BLAST approach, as detailed in Karlin and Altschul (supra)and incorporated herein by reference, searches for matches between aquery sequence and a database sequence, evaluates the statisticalsignificance of any matches found, and reports only those matches whichsatisfy the user-selected threshold of significance. In thisapplication, threshold was set at 10⁻²⁵ for nucleotides and 10⁻¹⁴ forpeptides. Incyte nucleotide sequences were searched against the GenBankdatabases for primate (pri), rodent (rod), and mammalian sequences(mam), and deduced amino acid sequences from the same clones aresearched against GenBank functional protein databases, mammalian (mamp),vertebrate (vrtp) and eukaryote (eukp), for homology.

[0184] IV Northern Analysis

[0185] Northern analysis is a laboratory technique used to detect thepresence of a transcript of a gene and involves the hybridization of alabeled nucleotide sequence to a membrane on which RNAs from aparticular cell type or tissue have been bound (Sambrook et al., supra).

[0186] Analogous computer techniques using BLAST (Altschul, S. F. 1993and 1990, supra) are used to search for identical or related moleculesin nucleotide databases such as GenBank or the LIFESEQ database (IncytePharmaceuticals). This analysis is much faster than multiple,membrane-based hybridizations. In addition, the sensitivity of thecomputer search can be modified to determine whether any particularmatch is categorized as exact or homologous.

[0187] The basis of the search is the product score which is defined as:

% sequence identity×% maximum BLAST score/100

[0188] The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1-2% error; and at 70, the match will be exact. Homologous moleculesare usually identified by selecting those which show product scoresbetween 15 and 40, although lower scores may identify related molecules.

[0189] The results of northern analysis are reported as a list oflibraries in which the transcript encoding LGFV occurs. Abundance andpercent abundance are also reported. Abundance directly reflects thenumber of times a particular transcript is represented in a cDNAlibrary, and percent abundance is abundance divided by the total numberof sequences examined in the cDNA library.

[0190] V Extension of LGFV-Encoding Polynucleotides to Full Length or toRecover Regulatory Sequences

[0191] Full length LGFV-encoding nucleic acid sequence (SEQ ID NO:2) isused to design oligonucleotide primers for extending a partialnucleotide sequence to full length or for obtaining 5′ or 3′, intron orother control sequences from genomic libraries. One primer issynthesized to initiate extension in the antisense direction (XLR) andthe other is synthesized to extend sequence in the sense direction(XLF). Primers are used to facilitate the extension of the knownsequence “outward” generating amplicons containing new, unknownnucleotide sequence for the region of interest. The initial primers aredesigned from the cDNA using OLIGO 4.06 (National Biosciences), oranother appropriate program, to be 22-30 nucleotides in length, to havea GC content of 50% or more, and to anneal to the target sequence attemperatures about 68°-72° C. Any stretch of nucleotides which wouldresult in hairpin structures and primer-primer dimerizations is avoided.

[0192] The original, selected cDNA libraries, or a human genomic libraryare used to extend the sequence; the latter is most useful to obtain 5′upstream regions. If more extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

[0193] By following the instructions for the XL-PCR kit (Perkin Elmer)and thoroughly mixing the enzyme and reaction mix, high fidelityamplification is obtained. Beginning with 40 pmol of each primer and therecommended concentrations of all other components of the kit, PCR isperformed using the Peltier Thermal Cycler (PTC200; M.J. Research,Watertown, Mass.) and the following parameters: Step 1 94° C. for 1 min(initial denaturation) Step 2 65° C. for 1 min Step 3 68° C. for 6 minStep 4 94° C. for 15 sec Step 5 65° C. for 1 min Step 6 68° C. for 7 minStep 7 Repeat step 4-6 for 15 additional cycles Step 8 94° C. for 15 secStep 9 65° C. for 1 min Step 10 68° C. for 7:15 min Step 11 Repeat step8-10 for 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (and holding)

[0194] A 5-10 μl aliquot of the reaction mixture is analyzed byelectrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gelto determine which reactions were successful in extending the sequence.Bands thought to contain the largest products are selected and removedfrom the gel. Further purification involves using a commercial gelextraction method such as QIAQUICK (QIAGEN Inc., Chatsworth, Calif.).After recovery of the DNA, Klenow enzyme is used to trimsingle-stranded, nucleotide overhangs creating blunt ends whichfacilitate religation and cloning.

[0195] After ethanol precipitation, the products are redissolved in 13μl of ligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase are added, and the mixture is incubated at roomtemperature for 2-3 hours or overnight at 16° C. Competent E. coli cells(in 40 μl of appropriate media) are transformed with 3 μl of ligationmixture and cultured in 80 μl of SOC medium (Sambrook et al., supra).After incubation for one hour at 37° C., the whole transformationmixture is plated on Luria Bertani (LB)-agar (Sambrook et al., supra)containing 2× Carb. The following day, several colonies are randomlypicked from each plate and cultured in 150μl of liquid LB/2× Carb mediumplaced in an individual well of an appropriate, commercially-available,sterile 96-well microtiter plate. The following day, 5 μl of eachovernight culture is transferred into a non-sterile 96-well plate andafter dilution 1:10 with water, 5 μl of each sample is transferred intoa PCR array.

[0196] For PCR amplification, 18 μl of concentrated PCR reaction mix(3.3×) containing 4 units of rTth DNA polymerase, a vector primer, andone or both of the gene specific primers used for the extension reactionare added to each well. Amplification is performed using the followingconditions: Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55°C. for 30 sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2-4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

[0197] Aliquots of the PCR reactions are run on agarose gels togetherwith molecular weight markers. The sizes of the PCR products arecompared to the original partial cDNAs, and appropriate clones areselected, ligated into plasmid, and sequenced.

[0198] VI Labeling and Use of Hybridization Probes

[0199] Hybridization probes derived from SEQ ID NO:2 are employed toscreen cDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base-pairs, is specificallydescribed, essentially the same procedure is used with larger cDNAfragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmolof each oligomer and 250 μCi of [γ-³²P] adenosine triphosphate(Amersham) and T4 polynucleotide kinase (DuPont NEN®, Boston, Mass.).The labeled oligonucleotides are substantially purified with SephadexG-25 superfine resin column (Pharmacia & Upjohn). A portion containing10⁷ counts per minute of each of the sense and antisenseoligonucleotides is used in a typical membrane based hybridizationanalysis of human genomic DNA digested with one of the followingendonucleases (Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II; DuPontNEN®).

[0200] The DNA from each digest is fractionated on a 0.7 percent agarosegel and transferred to nylon membranes (Nytran Plus, Schleicher &Schuell, Durham, N.H.). Hybridization is carried out for 16 hours at 40°C. To remove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots, or the blots areexposed in a PhosphorImager cassette (Molecular Dynamics, Sunnyvale,Calif.), hybridization patterns are compared visually.

[0201] VII Antisense Molecules

[0202] Antisense molecules to the LGFV-encoding sequence, or any partthereof, is used to inhibit in vivo or in vitro expression of naturallyoccurring LGFV. Although use of antisense oligonucleotides, comprisingabout 20 base-pairs, is specifically described, essentially the sameprocedure is used with larger cDNA fragments. An oligonucleotide basedon the coding sequences of LGFV, as shown in FIGS. 1A, 1B, and 1C, isused to inhibit expression of naturally occurring LGFV. Thecomplementary oligonucleotide is designed from the most unique 5′sequence as shown in FIGS. 1A, 1B, and 1C and used either to inhibittranscription by preventing promoter binding to the upstreamnontranslated sequence or translation of an LGFV-encoding transcript bypreventing the ribosome from binding. Using an appropriate portion ofthe signal and 5′ sequence of SEQ ID NO:2, an effective antisenseoligonucleotide includes any 15-20 nucleotides spanning the region whichtranslates into the signal or 5′ coding sequence of the polypeptide asshown in FIGS. 1A, 1B, and 1C.

[0203] VIII Expression of LGFV

[0204] Expression of LGFV is accomplished by subcloning the cDNAs intoappropriate vectors and transforming the vectors into host cells. Inthis case, the cloning vector, pSport, previously used for thegeneration of the cDNA library is used to express LGFV in E. coli.Upstream of the cloning site, this vector contains a promoter forβ-galactosidase, followed by sequence containing the amino-terminal Met,and the subsequent seven residues of β-galactosidase. Immediatelyfollowing these eight residues is a bacteriophage promoter useful fortranscription and a linker containing a number of unique restrictionsites.

[0205] Induction of an isolated, transformed bacterial strain with IPTGusing standard methods produces a fusion protein which consists of thefirst eight residues of β-galactosidase, about 5 to 15 residues oflinker, and the full length protein. The signal residues direct thesecretion of LGFV into the bacterial growth media which can be useddirectly in the following assay for activity.

[0206] IX Demonstration of LGFV Activity

[0207] Purified LGFV, or a vector for expressing the protein, can beadded to a mammalian cell culture using electroporation, liposometechnology or other methods well known in the art. The presence of thiscell cycle enhancer will increase the number of cell going through thecell cycle. Increased cell division/proliferation can be monitored inliquid culture by noting increases in cell number/ml of culture mediausing densitometry. Unstimulated vs stimulated cells will have differentdoubling times. Alternatively the mitotic index of control and treatedsubcultures may be compared using phase contrast microscopy.

[0208] X Production of LGFV Specific Antibodies

[0209] LGFV that is substantially purified using PAGE electrophoresis(Sambrook, supra), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols. The aminoacid sequence deduced from SEQ ID NO:2 is analyzed using DNASTARsoftware (DNASTAR Inc) to determine regions of high immunogenicity and acorresponding oligopolypeptide is synthesized and used to raiseantibodies by means known to those of skill in the art. Selection ofappropriate epitopes, such as those near the C-terminus or inhydrophilic regions, is described by Ausubel et al. (supra), and others.

[0210] Typically, the oligopeptides are 15 residues in length,synthesized using an Applied Biosystems Peptide Synthesizer Model 431Ausing fmoc-chemistry, and coupled to keyhole limpet hemocyanin (KLH,Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS; Ausubel et al.,supra). Rabbits are immunized with the oligopeptide-KLH complex incomplete Freund's adjuvant. The resulting antisera are tested forantipeptide activity, for example, by binding the peptide to plastic,blocking with 1% BSA, reacting with rabbit antisera, washing, andreacting with radioiodinated, goat anti-rabbit IgG.

[0211] XI Purification of Naturally Occurring LGFV Using SpecificAntibodies

[0212] Naturally occurring or recombinant LGFV is substantially purifiedby immunoaffinity chromatography using antibodies specific for LGFV. Animmunoaffinity column is constructed by covalently coupling LGFVantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

[0213] Media containing LGFV is passed over the immunoaffinity column,and the column is washed under conditions that allow the preferentialabsorbance of LGFV (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/LGFV binding (e.g., a buffer of pH 2-3 or a high concentrationof a chaotrope, such as urea or thiocyanate ion), and LGFV is collected.

[0214] XII Identification of Molecules Which Interact with LGFV

[0215] LGFV or biologically active fragments thereof are labeled with¹²⁵I Bolton-Hunter reagent (Bolton et al. (1973) Biochem. J. 133: 529).Candidate molecules previously arrayed in the wells of a multi-wellplate are incubated with the labeled LGFV, washed and any wells withlabeled LGFV complex are assayed. Data obtained using differentconcentrations of LGFV are used to calculate values for the number,affinity, and association of LGFV with the candidate molecules.

[0216] All publications and patents mentioned in the above specificationare herein incorporated by reference. Various modifications andvariations of the described method and system of the invention will beapparent to those skilled in the art without departing from the scopeand spirit of the invention. Although the invention has been describedin connection with specific preferred embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Indeed, various modifications of thedescribed modes for carrying out the invention which are obvious tothose skilled in molecular biology or related fields are intended to bewithin the scope of the following claims.

1 5 235 amino acids amino acid single linear lungast01 876242 1 Met ProHis Ala Phe Lys Pro Gly Asp Leu Val Phe Ala Lys Met Lys 1 5 10 15 GlyTyr Pro His Trp Pro Ala Arg Ile Asp Asp Ile Ala Asp Gly Ala 20 25 30 ValLys Pro Pro Pro Asn Lys Tyr Pro Ile Phe Phe Phe Gly Thr His 35 40 45 GluThr Ala Phe Leu Gly Pro Lys Asp Leu Phe Pro Tyr Glu Glu Ser 50 55 60 LysGlu Lys Phe Gly Lys Pro Asn Lys Arg Lys Gly Phe Ser Glu Gly 65 70 75 80Leu Trp Glu Ile Glu Asn Asn Pro Thr Val Lys Ala Ser Gly Tyr Gln 85 90 95Ser Ser Gln Lys Lys Ser Cys Val Glu Glu Pro Glu Pro Glu Pro Glu 100 105110 Ala Ala Glu Gly Asp Gly Asp Lys Lys Gly Asn Ala Glu Gly Ser Ser 115120 125 Asp Glu Glu Gly Lys Leu Val Ile Asp Glu Pro Ala Lys Glu Lys Asn130 135 140 Glu Lys Gly Ala Leu Lys Arg Arg Ala Gly Asp Leu Leu Glu AspSer 145 150 155 160 Pro Lys Arg Pro Lys Glu Ala Glu Asn Pro Glu Gly GluGlu Lys Glu 165 170 175 Ala Ala Thr Leu Glu Val Glu Arg Pro Leu Pro MetGlu Val Glu Lys 180 185 190 Asn Ser Thr Pro Ser Glu Pro Gly Ser Gly ArgGly Pro Pro Xaa Xaa 195 200 205 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Glu GluAla Thr Lys Glu Asp Ala 210 215 220 Glu Ala Pro Gly Ile Lys Ser His GluSer Leu 225 230 235 869 base pairs nucleic acid single linear lungast01876242 2 CCGCTGCAGC CGCTTTCTGC GGCCTGGGCC TCTCGCCGTC AGCATGCCACACGCCTTCAA 60 GCCCGGGGAC TTGGTGTTCG CTAAGATGAA GGGCTACCCT CACTGGCCTGCCAGGATCGA 120 CGACATCGCG GATGGCGCCG TGAAGCCCCC ACCCAACAAG TACCCCATCTTTTTCTTTGG 180 CACACACGAA ACAGCCTTCC TGGGCCCCAA AGACCTCTTC CCTTACGAGGAATCCAAGGA 240 GAAGTTTGGC AAGCCCAACA AGAGGAAAGG GTTCAGCGAG GGGCTGTGGGAGATCGAGAA 300 CAACCCTACT GTCAAGGCTT CCGGCTATCA GTCCTCCCAG AAAAAGAGCTGTGTGGAAGA 360 GCCTGAACCA GAGCCCGAAG CTGCAGAGGG TGACGGTGAT AAGAAGGGGAATGCAGAGGG 420 CAGCAGCGAC GAGGAAGGGA AGCTGGTCAT TGATGAGCCA GCCAAGGAGAAGAACGAGAA 480 AGGAGCGTTG AAGAGGAGAG CAGGGGACTT GCTGGAGGAC TCTCCTAAACGTCCCAAGGA 540 GGCAGAAAAC CCTGAAGGAG AGGAGAAGGA GGCAGCCACC TTGGAGGTTGAGAGGCCCCT 600 TCCTATGGAG GTGGAAAAGA ATAGCACCCC CTCTGAGCCC GGCTCTGGCCGGGGGCCTCC 660 CCNNNNNNNN NNNNNNNNNN NNNNNNNNNA GGAAGAGGCT ACCAAGGAAGATGCTGAGGC 720 CCCAGGCATC AAGAGTCATG AGAGCCTGTA GCCACCAATG TTTCAAGAGGAGCCCCCACC 780 CTGTTCCTGC TGCTGTCTGG GTGCTACTGG GGAAACTGGC CATGGGCTGCAAACTGGGNA 840 CCCCTTTTCC ANCNCAANCT GNTNTTCTT 869 240 amino acids aminoacid single linear GenBank 598956 3 Met Ser Arg Ser Asn Arg Gln Lys GluTyr Lys Cys Gly Asp Leu Val 1 5 10 15 Phe Ala Lys Met Lys Gly Tyr ProHis Trp Pro Ala Arg Ile Asp Glu 20 25 30 Met Pro Glu Ala Ala Val Lys SerThr Ala Asn Lys Tyr Gln Val Phe 35 40 45 Phe Phe Gly Thr His Glu Thr AlaPhe Leu Gly Pro Lys Asp Leu Phe 50 55 60 Pro Tyr Glu Glu Ser Lys Glu LysPhe Gly Lys Pro Asn Lys Arg Lys 65 70 75 80 Gly Phe Ser Glu Gly Leu TrpGlu Ile Glu Asn Asn Pro Thr Val Lys 85 90 95 Ala Ser Gly Tyr Gln Ser SerGln Lys Lys Ser Cys Val Glu Glu Pro 100 105 110 Glu Pro Glu Pro Glu AlaAla Glu Gly Asp Gly Asp Lys Lys Gly Asn 115 120 125 Ala Glu Gly Ser SerAsp Glu Glu Gly Lys Leu Val Ile Asp Glu Pro 130 135 140 Ala Lys Glu LysAsn Glu Lys Gly Ala Leu Lys Arg Arg Ala Gly Asp 145 150 155 160 Leu LeuGlu Asp Ser Pro Lys Arg Pro Lys Glu Ala Glu Asn Pro Glu 165 170 175 GlyGlu Glu Lys Glu Ala Ala Thr Leu Glu Val Glu Arg Pro Leu Pro 180 185 190Met Glu Val Glu Lys Asn Ser Thr Pro Ser Glu Pro Gly Ser Gly Arg 195 200205 Gly Pro Pro Gln Glu Glu Glu Glu Glu Glu Asp Glu Glu Glu Glu Ala 210215 220 Thr Lys Glu Asp Ala Glu Ala Pro Gly Ile Arg Asp His Glu Ser Leu225 230 235 240 2376 base pairs nucleic acid single linear GenBank598956 4 GAGGAGGAGT GGGGACCGGG CGGGGGGTGG AGGAAGAGGC CTCGCGCAGAGGAGGGAGCA 60 ATTGAATTTC AAACACAAAC AACTCGACGA GCGCGCACCC ACCGCGCCGGAGCCTTGCCC 120 CGATCCGCGC CCGCCCCGTC CGTGCGGCGC GCGGGCGGAG ACGCCGTGGCCGCGCCGGAG 180 CTCGGGCCGG GGGCCACCAT CGAGGCGGGG GCCGCGCGAG GGCCGGAGCGGAGCGGCGCC 240 GCCACCGCCG CACGCGCAAA CTTGGGCTCG CGCTTCCCGG CCCGGCGCGGAGCCCGGGGC 300 GCCCGGAGCC CCGCCATGTC GCGATCCAAC CGGCAGAAGG AGTACAAATGCGGGGACCTG 360 GTGTTCGCCA AGATGAAGGG CTACCCACAC TGGCCGGCCC GGATTGACGAGATGCCTGAG 420 GCTGCCGTGA AATCAACAGC CAACAAATAC CAAGTCTTTT TTTTCGGGACCCACGAGACG 480 GCATTCCTGG GCCCCAAAGA CCTCTTCCCT TACGAGGAAT CCAAGGAGAAGTTTGGCAAG 540 CCCAACAAGA GGAAAGGGTT CAGCGAGGGG CTGTGGGAGA TCGAGAACAACCCTACTGTC 600 AAGGCTTCCG GCTATCAGTC CTCCCAGAAA AAGAGCTGTG TGGAAGAGCCTGAACCAGAG 660 CCCGAAGCTG CAGAGGGTGA CGGTGATAAG AAGGGGAATG CAGAGGGCAGCAGCGACGAG 720 GAAGGGAAGC TGGTCATTGA TGAGCCAGCC AAGGAGAAGA ACGAGAAAGGAGCGTTGAAG 780 AGGAGAGCAG GGGACTTGCT GGAGGACTCT CCTAAACGTC CCAAGGAGGCAGAAAACCCT 840 GAAGGAGAGG AGAAGGAGGC AGCCACCTTG GAGGTTGAGA GGCCCCTTCCTATGGAGGTG 900 GAAAAGAATA GCACCCCCTC TGAGCCCGGC TCTGGCCGGG GGCCTCCCCAAGAGGAAGAA 960 GAAGAGGAGG ATGAAGAGGA AGAGGCTACC AAGGAAGATG CTGAGGCCCCAGGCATCAGA 1020 GATCATGAGA GCCTGTAGCC ACCAATGTTT CAAGAGGAGC CCCCACCCTGTTCCTGCTGC 1080 TGTCTGGGTG CTACTGGGGA AACTGGCCAT GGCCTGCAAA CTGGGAACCCCTTTCCCACC 1140 CCAACCTGCT CTCCTCTTCT ACTCACTTTT CCCACTCCAA GCCCAGCCCATGGAGATTGA 1200 CCTGGATGGG GCAGGCCACC TGGCTCTCAC CTCTAGGTCC CCATACTCCTATGATCTGAG 1260 TCAGAGCCAT GTCTTCTCCC TGGAATGAGT TGAGGCCACT GTGTTCCTTCCGCTTGGAGC 1320 TATTTTCCAG GCTTCTGCTG GGGCCTGGGA CAACTGCTCC CACCTCCTGACACCCTTCTC 1380 CCACTCTCCT AGGCATTCTG GACCTCTGGG TTGGGATCAG GGGTAGGAATGGAAGGATGG 1440 AGCATCAACA GCAGGGTGGG CTTGTGGGGC CTGGGAGGGG CAATCCTCAAATGCGGGGTG 1500 GGGGCAGCAC AGGAGGGCGG CCTCCTTCTG AGCTCCTGTC CCCTGCTACACCTATTATCC 1560 CAGCTGCCTA GATTCAGGGA AAGTGGGACA GCTTGTAGGG GAGGGGCTCCTTTCCATAAA 1620 TCCTTGATGA TTGACAACAC CCATTTTTCC TTTTGCCGAC CCCAAGAGTTTTGGGAGTTG 1680 TAGTTAATCA TCAAGAGAAT TTGGGGCTTC CAAGTTGTTC GGGCCAAGGACCTGAGACCT 1740 GAAGGGTTGA CTTTACCCAT TTGGGTGGGA GTGTTGAGCA TCTGTCCCCCTTTAGATCTC 1800 TGAAGCCACA AATAGGATGC TTGGGAAGAC TCCTAGCTGT CCTTTTTCCTCTCCACACAG 1860 TGCTCAAGGC CAGCTTATAG TCATATATAT CACCCAGACA TAAAGGAAAAGACACATTTT 1920 TTAGGAAATG TTTTTAATAA AAGAAAATTA CAAAAAAAAA TTTTAAAGACCCCTAACCCT 1980 TTGTGTGCTC TCCATTCTGC TCCTTCCCCA TCGTTGCCCC CATTTCTGAGGTGCACTGGG 2040 AGGCTCCCCT TCTATTTGGG GCTTGATGAC TTTCTTTTTG TAGCTGGGGCTTTGATGTTC 2100 CTTCCAGTGT CATTTCTCAT CCACATACCC TGACCTGGCC CCCTCAGTGTTGTCACCAGA 2160 TCTGATTTGT AACCCACTGA GAGGACAGAG AGAAATAAGT GCCCTCTCCCACCCTCTTCC 2220 TACTGGTCTC TCTATGCCTC TCTACAGTCT CGTCTCTTTT ACCCTGGCCCCTCTCCCTTG 2280 GGCTCTGATG AAAAATTGCT GACTGTAGCT TTGGAAGTTT AGCTCTGAGAACCGTAGATG 2340 ATTTCAGTTC TAGGAAAATA AAACCCGTTG ATTACT 2376 237 aminoacids amino acid single linear GenBank 945419 5 Met Ser Arg Ser Asn ArgGln Lys Glu Tyr Lys Cys Gly Asp Leu Val 1 5 10 15 Phe Ala Lys Met LysGly Tyr Pro His Trp Pro Ala Arg Ile Asp Glu 20 25 30 Met Pro Glu Ala AlaVal Lys Ser Thr Ala Asn Lys Tyr Gln Val Phe 35 40 45 Phe Phe Gly Thr HisGlu Thr Ala Phe Leu Gly Pro Lys Asp Leu Phe 50 55 60 Pro Tyr Glu Glu SerLys Glu Lys Phe Gly Lys Pro Asn Lys Arg Lys 65 70 75 80 Gly Phe Ser GluGly Leu Trp Glu Ile Glu Asn Asn Pro Thr Val Lys 85 90 95 Ala Ser Gly TyrGln Ser Ser Gln Lys Lys Ser Cys Ala Ala Glu Pro 100 105 110 Glu Val GluPro Glu Ala His Glu Gly Asp Gly Asp Lys Lys Gly Ser 115 120 125 Ala GluGly Ser Ser Asp Glu Glu Gly Lys Leu Val Ile Asp Glu Pro 130 135 140 AlaLys Glu Lys Asn Glu Lys Gly Thr Leu Lys Arg Arg Ala Gly Asp 145 150 155160 Val Leu Glu Asp Ser Pro Lys Arg Pro Lys Glu Ser Gly Asp His Glu 165170 175 Glu Glu Asp Lys Glu Ile Ala Ala Leu Glu Gly Glu Arg His Leu Pro180 185 190 Val Glu Val Glu Lys Asn Ser Thr Pro Ser Glu Pro Asp Ser GlyGln 195 200 205 Gly Pro Pro Ala Glu Glu Glu Glu Gly Glu Glu Glu Ala AlaLys Glu 210 215 220 Glu Ala Glu Ala Pro Gly Val Arg Asp His Glu Ser Leu225 230 235

What is claimed is:
 1. An isolated polypeptide comprising an amino acidsequence selected from the group consisting of: a) a polypeptidecomprising an amino acid sequence of SEQ ID NO:1, b) a naturallyoccurring polypeptide comprising an amino acid sequence at least 90%identical to an amino acid sequence of SEQ ID NO:1, c) a biologicallyactive fragment of a polypeptide having an amino acid sequence of SEQ IDNO:1, and d) an immunogenic fragment of a polypeptide having an aminoacid sequence of SEQ ID NO:1.
 2. An isolated polypeptide of claim 1,having a sequence of SEQ ID NO:1.
 3. An isolated polynucleotide encodinga polypeptide of claim
 1. 4. A recombinant polynucleotide comprising apromoter sequence operably linked to a polynucleotide of claim
 3. 5. Acell transformed with a recombinant polynucleotide of claim
 4. 6. Atransgenic organism comprising a recombinant polynucleotide of claim 4.7. A method for producing a polypeptide of claim 1, the methodcomprising: a) culturing a cell under conditions suitable for expressionof the polypeptide, wherein said cell is transformed with a recombinantpolynucleotide, and said recombinant polynucleotide comprises a promotersequence operably linked to a polynucleotide encoding the polypeptide ofclaim 1, and b) recovering the polypeptide so expressed.
 8. An isolatedantibody which specifically binds to a polypeptide of claim
 1. 9. Anisolated polynucleotide comprising a sequence selected from the groupconsisting of: a) a polynucleotide comprising a polynucleotide sequenceof SEQ ID NO:2, b) a naturally occurring polynucleotide comprising apolynucleotide sequence at least 90% identical to a polynucleotidesequence of SEQ ID NO:2, c) a polynucleotide having a sequencecomplementary to a polynucleotide of a), d) a polynucleotide having asequence complementary to a polynucleotide of b) and p1 e) an RNAequivalent of a)-d).
 10. An isolated polynucleotide comprising at least60 contiguous nucleotides of a polynucleotide of claim
 9. 11. A methodfor detecting a target polynucleotide in a sample, said targetpolynucleotide having a sequence of a polynucleotide of claim 9, themethod comprising: a) hybridizing the sample with a probe comprising atleast 20 contiguous nucleotides comprising a sequence complementary tosaid target polynucleotide in the sample, and which probe specificallyhybridizes to said target polynucleotide, under conditions whereby ahybridization complex is formed between said probe and said targetpolynucleotide or fragments thereof, and b) detecting the presence orabsence of said hybridization complex, and, optionally, if present, theamount thereof.
 12. A method of claim 11, wherein the probe comprises atleast 60 contiguous nucleotides.
 13. A method for detecting a targetpolynucleotide in a sample, said target polynucleotide having a sequenceof a polynucleotide of claim 9, the method comprising: a) amplifyingsaid target polynucleotide or fragment thereof using polymerase chainreaction amplification, and b) detecting the presence or absence of saidamplified target polynucleotide or fragment thereof, and, optionally, ifpresent, the amount thereof.
 14. A composition comprising a polypeptideof claim 1 and a pharmaceutically acceptable excipient.
 15. Acomposition of claim 14, wherein the polypeptide has an amino acidsequence of SEQ ID NO:1.
 16. A method for treating a disease orcondition associated with decreased expression of functional LGFV,comprising administering to a patient in need of such treatment thecomposition of claim
 14. 17. A method for screening a compound foreffectiveness as an agonist of a polypeptide of claim 1, the methodcomprising: a) exposing a sample comprising a polypeptide of claim 1 toa compound, and b) detecting agonist activity in the sample.
 18. Acomposition comprising an agonist compound identified by a method ofclaim 17 and a pharmaceutically acceptable excipient.
 19. A method fortreating a disease or condition associated with decreased expression offunctional LGFV, comprising administering to a patient in need of suchtreatment a composition of claim
 18. 20. A method for screening acompound for effectiveness as an antagonist of a polypeptide of claim 1,the method comprising: a) exposing a sample comprising a polypeptide ofclaim 1 to a compound, and b) detecting antagonist activity in thesample.
 21. A composition comprising an antagonist compound identifiedby a method of claim 20 and a pharmaceutically acceptable excipient. 22.A method for treating a disease or condition associated withoverexpression of functional LGFV, comprising administering to a patientin need of such treatment a composition of claim
 21. 23. A method ofscreening for a compound that specifically binds to the polypeptide ofclaim 1, said method comprising the steps of: a) combining thepolypeptide of claim 1 with at least one test compound under suitableconditions, and b) detecting binding of the polypeptide of claim 1 tothe test compound, thereby identifying a compound that specificallybinds to the polypeptide of claim
 1. 24. A method of screening for acompound that modulates the activity of the polypeptide of claim 1, saidmethod comprising: a) combining the polypeptide of claim 1 with at leastone test compound under conditions permissive for the activity of thepolypeptide of claim 1, b) assessing the activity of the polypeptide ofclaim 1 in the presence of the test compound, and c) comparing theactivity of the polypeptide of claim 1 in the presence of the testcompound with the activity of the polypeptide of claim 1 in the absenceof the test compound, wherein a change in the activity of thepolypeptide of claim 1 in the presence of the test compound isindicative of a compound that modulates the activity of the polypeptideof claim
 1. 25. A method for screening a compound for effectiveness inaltering expression of a target polynucleotide, wherein said targetpolynucleotide comprises a polynucleotide sequence of SEQ ID NO:2, themethod comprising: a) exposing a sample comprising the targetpolynucleotide to a compound, under conditions suitable for theexpression of the target polynucleotide, b) detecting altered expressionof the target polynucleotide, and c) comparing the expression of thetarget polynucleotide in the presence of varying amounts of the compoundand in the absence of the compound.
 26. A method for assessing toxicityof a test compound, said method comprising: a) treating a biologicalsample containing nucleic acids with the test compound; b) hybridizingthe nucleic acids of the treated biological sample with a probecomprising at least 20 contiguous nucleotides of a polynucleotide ofclaim 9 under conditions whereby a specific hybridization complex isformed between said probe and a target polynucleotide in the biologicalsample, said target polynucleotide comprising a polynucleotide sequenceof a polynucleotide of claim 9 or fragment thereof; c) quantifying theamount of hybridization complex; and d) comparing the amount ofhybridization complex in the treated biological sample with the amountof hybridization complex in an untreated biological sample, wherein adifference in the amount of hybridization complex in the treatedbiological sample is indicative of toxicity of the test compound.
 27. Adiagnostic test for a condition or disease associated with theexpression of LGFV in a biological sample comprising the steps of: a)combining the biological sample with an antibody of claim 8, underconditions suitable for the antibody to bind the polypeptide and form anantibody:polypeptide complex; and b) detecting the complex, wherein thepresence of the complex correlates with the presence of the polypeptidein the biological sample.
 28. The antibody of claim 8, wherein theantibody is: a) a chimeric antibody, b) a single chain antibody, c) aFab fragment, d) a F(ab′)₂ fragment, or e) a humanized antibody.
 29. Acomposition comprising an antibody of claim 8 and an acceptableexcipient.
 30. A method of diagnosing a condition or disease associatedwith the expression of LGFV in a subject, comprising administering tosaid subject an effective amount of the composition of claim
 29. 31. Acomposition of claim 29, wherein the antibody is labeled.
 32. A methodof diagnosing a condition or disease associated with the expression ofLGFV in a subject, comprising administering to said subject an effectiveamount of the composition of claim
 31. 33. A method of preparing apolyclonal antibody with the specificity of the antibody of claim 8comprising: a) immunizing an animal with a polypeptide having an aminoacid sequence of SEQ ID NO:1, or an immunogenic fragment thereof, underconditions to elicit an antibody response; b) isolating antibodies fromsaid animal; and c) screening the isolated antibodies with thepolypeptide, thereby identifying a polyclonal antibody which bindsspecifically to a polypeptide having an amino acid sequence of SEQ IDNO:1.
 34. An antibody produced by a method of claim
 33. 35. Acomposition comprising the antibody of claim 34 and a suitable carrier.36. A method of making a monoclonal antibody with the specificity of theantibody of claim 8 comprising: a) immunizing an animal with apolypeptide having an amino acid sequence of SEQ ID NO:1, or animmunogenic fragment thereof, under conditions to elicit an antibodyresponse; b) isolating antibody producing cells from the animal; c)fusing the antibody producing cells with immortalized cells to formmonoclonal antibody-producing hybridoma cells; d) culturing thehybridoma cells; and e) isolating from the culture monoclonal antibodywhich binds specifically to a polypeptide having an amino acid sequenceof SEQ ID NO:1.
 37. A monoclonal antibody produced by a method of claim36.
 38. A composition comprising the antibody of claim 37 and a suitablecarrier.
 39. The antibody of claim 8, wherein the antibody is producedby screening a Fab expression library.
 40. The antibody of claim 8,wherein the antibody is produced by screening a recombinantimmunoglobulin library.
 41. A method for detecting a polypeptide havingan amino acid sequence of SEQ ID NO:1 in a sample, comprising the stepsof: a) incubating the antibody of claim 8 with a sample under conditionsto allow specific binding of the antibody and the polypeptide; and b)detecting specific binding, wherein specific binding indicates thepresence of a polypeptide having an amino acid sequence of SEQ ID NO:1in the sample.
 42. A method of purifying a polypeptide having an aminoacid sequence of SEQ ID NO:1 from a sample, the method comprising: a)incubating the antibody of claim 8 with a sample under conditions toallow specific binding of the antibody and the polypeptide; and b)separating the antibody from the sample and obtaining the purifiedpolypeptide having an amino acid sequence of SEQ ID NO:1.
 43. Amicroarray wherein at least one element of the microarray is apolynucleotide of claim
 10. 44. A method for generating a transcriptimage of a sample which contains polynucleotides, the method comprisingthe steps of: a) labeling the polynucleotides of the sample, b)contacting the elements of the microarray of claim 43 with the labeledpolynucleotides of the sample under conditions suitable for theformation of a hybridization complex, and c) quantifying the expressionof the polynucleotides in the sample.
 45. An array comprising differentnucleotide molecules affixed in distinct physical locations on a solidsubstrate, wherein at least one of said nucleotide molecules comprises afirst oligonucleotide or polynucleotide sequence specificallyhybridizable with at least 30 contiguous nucleotides of a targetpolynucleotide, said target polynucleotide having a sequence of claim 9.46. An array of claim 45, wherein said first oligonucleotide orpolynucleotide sequence is completely complementary to at least 30contiguous nucleotides of said target polynucleotide.
 47. An array ofclaim 45, wherein said first oligonucleotide or polynucleotide sequenceis completely complementary to at least 60 contiguous nucleotides ofsaid target polynucleotide.
 48. An array of claim 45, which is amicroarray.
 49. An array of claim 45, further comprising said targetpolynucleotide hybridized to said first oligonucleotide orpolynucleotide.
 50. An array of claim 45, wherein a linker joins atleast one of said nucleotide molecules to said solid substrate.
 51. Anarray of claim 45, wherein each distinct physical location on thesubstrate contains multiple nucleotide molecules having the samesequence, and each distinct physical location on the substrate containsnucleotide molecules having a sequence which differs from the sequenceof nucleotide molecules at another physical location on the substrate.